Compositions, methods and kits to detect adenovirus nucleic acids

ABSTRACT

The disclosed invention is related to methods, compositions, kits and isolated nucleic acid sequences for targeting Adenovirus nucleic acid. Compositions include amplification oligomers and/or detection probe oligomers. Kits and methods comprise at least one of these oligomers.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/253,819, filed Oct. 5, 2011, now issued as U.S. Pat. No. 8,715,939,the contents of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to the detection of infectious agents,more specifically to the detection of Adenovirus. Compositions, methodsand kits are described for the detection of Adenovirus by using in vitronucleic acid amplification techniques.

Introduction

Adenovirus may cause infections in a number of different organsincluding the gastrointestinal tract, the upper respiratory tract andthe eyes. In individuals with a properly functioning immune system,Adenovirus infections are not typically associated with life-threateningdisease. However, Adenovirus can cause serious infection inimmunocompromised patients—such as HIV-positive individuals and inpatients receiving bone marrow transplants. More than 50 different humanAdenovirus serotypes have been identified. On the basis of variousproperties of Adenovirus, they have been divided into six majorsubgroups (subgenera or species A-F), with recent literature pointingtowards a the presence of a seventh serotype.

Early approaches for detecting Adenovirus detection relied mainly onserological tests and cell culture. In immunosuppressed patients,however, the use of serological tests is limited due to the impairedimmune response, and evaluation of positive cultures is a relativelyslow method. The introduction of PCR-based assays has provided newmethods for the rapid, specific and sensitive detection of Adenovirus.Many of these diagnostic approaches, however, do not effectively coverall Adenovirus serotypes or use low stringency conditions to permitdetection of the genetically highly diverse adenoviruses.

The homology of adenovirus DNA sequences between different species islow. Even conserved regions within the Adenovirus genome display onlylimited homology between adenoviruses from different species. In manyinstances, considerable differences in DNA sequence even exist betweenserotypes belonging to the same species. These facts underscore thedifficulty to develop molecular tests that facilitate reliable screeningfor Adenovirus infections with the required broad specificity.

A molecular based assay is required to permit the sensitive and specificdetection of multiple adenovirus serotypes.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide methods,compositions and kits that can be used to specifically detect with highsensitivity Adenovirus nucleic acid. Advantageously, the methods,compositions and kits may be used to specifically detect with highsensitivity many (eg. 5 or more, 10 or more, 20 or more, 30 or more, 40or more or 50 or more), or all known serotypes of adenovirus.

In one aspect, there is provided a method for specifically detecting anAdenovirus target nucleic acid in a sample comprising the steps of: (a)contacting a sample suspected of containing at least an Adenovirusnucleic acid with at least two amplification oligomers for generating anamplicon, wherein each of said at least two amplification oligomers isfrom 10 to about 50 nucleotides in length and wherein the amplificationoligomers are respectively configured to specifically hybridize toregions within a target sequence of Adenovirus selected from the groupconsisting of from nucleotides 1 to 99 and from nucleotides 83 to 175 ofAccession Number AB330090.1 (SEQ ID No. 47); (b) providing conditionssufficient for generating an amplicon from an Adenovirus target nucleicacid present in said sample using said amplification oligomers from step(a); and (c) providing conditions for detecting said amplicon anddetermining whether an Adenovirus target nucleic acid is present in saidsample.

In one embodiment, at least one of said at least two amplificationoligomers comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NOS: 1to 9, 11 to 16, 25 to 28, 31 to 35, 38, and 42 to 46 or a combination oftwo or more thereof.

In one embodiment, at least one first amplification oligomer comprises,consists of consists essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NOS: 1, 5, 11, 12, 25, 26,31, 32, 33, 34, 35 or 38 or a combination of two or more thereof.

In one embodiment, at least one second amplification oligomer comprises,consists of consists essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NOS: 2, 3, 6, 7, 8, 9, 13,14, 15, 16, 27, 28, 42, 43, 44, 45 or 46 or a combination of two or morethereof.

In one embodiment, a first amplification oligomer comprises, consists ofconsists essentially of a target hybridizing sequence as set forth inSEQ ID NO:1 and at least one second amplification oligomer comprises,consists or consists essentially of SEQ ID NO: 2 and/or SEQ ID NO: 3.

In one embodiment, a first amplification oligomer comprises, consists ofconsists essentially of a target hybridizing sequence as set forth inSEQ ID NO:5 and at least one second amplification oligomer comprises,consists or consists essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NOS: 6 to 9 or acombination of two or more thereof.

In one embodiment, at least one first amplification oligomer comprises,consists or consists essentially of a target hybridizing sequence as setforth in SEQ ID NO:11 and/or 12 and at least one second amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NOS:13 to 16 or a combination of two or more thereof.

In one embodiment, at least one first amplification oligomer comprises,consists of consists essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:31 to 35 or 38 or acombination of two or more thereof and at least one second amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence as set forth in SEQ ID NO: 27 and/or 28.

In one embodiment, at least one first amplification oligomer comprises,consists of consists essentially of a target hybridizing sequence as setforth in SEQ ID NO:25 and/or 26 and at least one second amplificationoligomer comprises, consists of consists essentially of a targethybridizing sequence as set forth in SEQ ID NO: 27 and/or 28.

In one embodiment, at least two first amplification oligomers eachrespectively comprise, consist or consist essentially of the targethybridizing sequence as set forth in SEQ ID NO:25 and 26 and at leasttwo second amplification oligomers each respectively comprise, consistor consist essentially of a target hybridizing sequence as set forth inSEQ ID NO: 27 and 28.

In one embodiment, the combination of amplification oligomers is SEQ IDNos 25, 26, 27 and 28; or SEQ ID Nos 26, 27 and 28; or SEQ ID Nos 25, 26and 28; or SEQ ID Nos 25, 26 and 27; or SEQ ID Nos 25, 27 and 28; or SEQID Nos 25 and 27; or SEQ ID Nos 25 and 28; or SEQ ID Nos 26 and 27; orSEQ ID Nos 26 and 28.

In one embodiment, said detection step comprises contacting saidamplification product with at least one detection probe configured tohybridize to a portion of said amplification product.

In one embodiment, the detection probe comprises, consists of consistsessentially of a target hybridizing sequence selected from the groupconsisting of SEQ ID Nos 10, 17 to 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof.

In one embodiment, the detection probe comprises, consists of consistsessentially of a sequence as set forth in SEQ ID No. 36 and/or SEQ IDNo. 37.

In a further aspect, there is provided a method for detecting anAdenovirus nucleic acid comprising the steps of: (a) contacting a testsample comprising nucleic acid with at least one hybridization assayprobe comprising, consisting or consisting essentially of the sequenceselected from the group consisting of SEQ ID Nos. 4, 10, 17, 18, 19, 20,21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or a combination of two ormore thereof; and (b) determining if a probe:target duplex has formedunder stringent hybridization conditions in the test sample, wherein thepresence of the probe:target duplex is an indication of the presence ofAdenovirus nucleic acid in the test sample.

In a further aspect, there is provided a composition for use in anAdenovirus target nucleic acid amplification assay comprising at leasttwo amplification oligomers capable of stably hybridizing to Adenovirustarget nucleic acid, wherein each of said at least two amplificationoligomers is from 10 to about 50 nucleotides in length and wherein theamplification oligomers are respectively configured to specificallyhybridize to regions within a target sequence of Adenovirus selectedfrom the group consisting of from nucleotides 1 to 99 and fromnucleotides 83 to 175 of Accession Number AB330090.1 (SEQ ID No. 47).

At least one first amplification oligomer comprises, consists ofconsists essentially of a target hybridizing sequence as set forth inSEQ ID NO:25 and/or SEQ ID NO:26 and wherein at least one secondamplification oligomer comprises, consists of consists essentially of atarget hybridizing sequence as set forth in SEQ ID NO:27 and/or SEQ IDNO:28.

In one embodiment, the first and second amplification oligomers areselected from the group consisting of SEQ ID Nos 25, 26, 27 and 28; SEQID Nos 26, 27 and 28; SEQ ID Nos 25, 26 and 28; SEQ ID Nos 25, 26 and27; SEQ ID Nos 25, 27 and 28; SEQ ID Nos 25 and 27; SEQ ID Nos 25 and28; SEQ ID Nos 26 and 27 and SEQ ID Nos 26 and 28 or a combination oftwo or more thereof.

In one embodiment, the composition further comprises a detection probe.

In one embodiment, the detection probe comprises, consists of consistsessentially of a sequence selected from the group consisting of SEQ IDNos 10, 17 to 24, 29, 30, 36, 37 39 and 40 or a combination of two ormore thereof.

In one embodiment, the detection probe comprises, consists of consistsessentially of a sequence as set forth in SEQ ID No. 36 and/or SEQ IDNo. 37.

In a further aspect, there is provided a kit comprising the compositionof the present invention and optionally a set of instructions forperforming same.

In a further aspect, there is provided an isolated DNA sequencesubstantially corresponding to the sequence set forth in any of SEQ IDNos: 1 to 46 or the corresponding isolated RNA sequence.

In one embodiment, said sequence is the complement or the reversecomplement thereof.

In a further aspect, there is provided the use of the isolated DNAsequence for amplifying and/or detecting Adenovirus nucleic acid.

DETAILED DESCRIPTION

Nucleic acid oligomer sequences are disclosed that may serve as primersand/or detection probes for amplification and/or detection of Adenovirusnucleic acids. The Adenovirus nucleic acids may be detected in a sampleby using methods of in vitro nucleic acid amplification—such as PCR (eg.Taqman PCR)—or transcription-mediated amplification—such as TMA orNASBA. Probes for detection of the amplified nucleic acid sequences arealso described. Detection probes hybridize specifically to at least aportion of the amplified sequence, either after completion of or duringthe amplification process. Some embodiments detect the amplifiedproducts by using a homogeneous detection method that detects, in amixture, a labeled probe bound specifically to an amplified sequence(eg., see Arnold et al., 1989, Clin. Chem. 35:1588-1594; U.S. Pat. No.5,658,737, Nelson et al., and U.S. Pat. Nos. 5,118,801 and 5,312,728,Lizardi et al.). The methods may use oligonucleotide sequences thatserve as capture probes for processing a sample to capture the targetAdenovirus nucleic acid and separate it from other sample components(eg. see U.S. Pat. Nos. 6,110,678, 6,280,952 and 6,534,273).

Methods disclosed herein can be used to detect Adenovirus nucleic acidspresent in samples from or derived from animals and humans.

Compositions disclosed herein include amplification oligomers that canbe used to specifically amplify selected nucleic acid sequences presentin Adenovirus genomic sequences, and optionally nucleic acid probes fordetecting the amplified sequences.

The disclosed nucleic acid sequences and methods are useful foramplifying and detecting Adenovirus nucleic acids from or derived fromviral particles present in a sample in a relatively short time so thatdiagnosis can be made quickly and so effective treatment can beinitiated and spread of the virus limited. The methods are useful forscreening for individuals who have Adenovirus infections but who do notexhibit definitive symptoms and are particularly useful for screeningpatients who have a higher risk of death or serious complications fromAdenovirus infections, eg., the young, elderly, or immunocompromisedindividuals. The methods are also useful for rapid screening of manysamples. The methods are useful because they minimize the risk ofexposure of laboratory personnel to the infectious Adenovirus agents,thereby limiting the risk of infection and spread of the virus. Thus,the methods and compositions disclosed herein respond to a need forrapid, sensitive, and specific testing of clinical samples that maycontain Adenovirus.

The disclosed probe sequences may be used as primers, and the disclosedprimers may be used as probes. The same is true for the disclosed probedomains and primer domains. Thus, the probe domains disclosed herein maybe used as primer domains. Likewise, primer domains disclosed herein maybe used as probe domains.

The amplification oligomers disclosed herein are further contemplated ascomponents of multiplex amplification reactions wherein several ampliconspecies that can be produced from an assortment (eg. two or more, threeor more, for or more, five or more, six or more, or even ten or more) oftarget-specific primers. For example, it is contemplated that more thanone of the amplification systems disclosed herein can be combined toresult in a multiplex assay that is both robust and broad in itscapacity for target detection—such as the ability to detect 5 or more,10 or more, 20 or more, 30 or more, 40 or more or 50 or more, or allknown serotypes of adenovirus.

To aid in understanding aspects of the disclosure, some terms usedherein are described in more detail. All other scientific and technicalterms used herein have the same meaning as commonly understood by thoseskilled in the relevant art, such as may be provided in Dictionary ofMicrobiology and Molecular Biology, 2nd ed. (Singleton et al., 1994,John Wiley & Sons, New York, N.Y.), The Harper Collins Dictionary ofBiology (Hale & Marham, 1991, Harper Perennial, N.Y., N.Y.), andreferences cited herein. Unless mentioned otherwise, the techniquesemployed or contemplated herein are standard methods well known to aperson of ordinary skill in the art of molecular biology.

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “a nucleic acid,” is understood torepresent one or more nucleic acids. As such, the terms “a” (or “an”),“one or more,” and “at least one” can be used interchangeably herein.

Sample. A “sample” or “specimen”, including “biological” or “clinical”samples may contain or may be suspected of containing Adenovirus orcomponents thereof, such as nucleic acids or fragments of nucleic acids.A sample may be a complex mixture of components. Samples include“biological samples” which include any tissue or material derived from aliving or dead mammal or organism, including, for example, blood,plasma, serum, blood cells, saliva, mucous and cerebrospinal fluid.Samples may also include samples of in vitro cell culture constituentsincluding, eg., conditioned media resulting from the growth of cells andtissues in culture medium. The sample may be treated to physically ormechanically disrupt tissue or cell structure to release intracellularnucleic acids into a solution which may contain enzymes, buffers, salts,detergents and the like, to prepare the sample for analysis. In one stepof the methods described herein, a sample is provided that is suspectedof containing at least an Adenovirus target nucleic acid. Accordingly,this step excludes the physical step of obtaining the sample from asubject.

Nucleic Acid. This refers to a multimeric compound comprising two ormore covalently bonded nucleosides or nucleoside analogs havingnitrogenous heterocyclic bases, or base analogs, where the nucleosidesare linked together by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine, 5-methylisocytosine, isoguanine; TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992, Abraham et al., 2007, BioTechniques 43: 617-24), which includederivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” residues in whichthe backbone does not include a nitrogenous base for one or moreresidues (U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′ methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., 2004, Biochemistry 43(42):13233-41). Nucleic acids may includemodified bases to alter the function or behaviour of the nucleic acid,e.g., addition of a 3′-terminal dideoxynucleotide to block additionalnucleotides from being added to the nucleic acid. Synthetic methods formaking nucleic acids in vitro are well known in the art although nucleicacids may be purified from natural sources using routine techniques.

Polynucleotide. The term denotes a nucleic acid chain. Throughout thisapplication, nucleic acids are designated by the 5′-terminus to the3′-terminus. Standard nucleic acids, e.g., DNA and RNA, are typicallysynthesized “3′-to-5′,” i.e., by the addition of nucleotides to the5′-terminus of a growing nucleic acid.

Nucleotide. This is a subunit of a nucleic acid consisting of aphosphate group, a 5-carbon sugar and a nitrogenous base. The 5-carbonsugar found in RNA is ribose. In DNA, the 5-carbon sugar is2′-deoxyribose. The term also includes analogs of such subunits, such asa methoxy group at the 2′ position of the ribose (2′-O-Me, or 2′methoxy). As used herein, methoxy oligonucleotides containing “T”residues have a methoxy group at the 2′ position of the ribose moiety,and a uracil at the base position of the nucleotide.

Non-nucleotide unit. This is a unit that does not significantlyparticipate in hybridization of a polymer. Such units must not, forexample, participate in any significant hydrogen bonding with anucleotide, and would exclude units having as a component one of thefive nucleotide bases or analogs thereof.

Target nucleic acid. This is a nucleic acid comprising a “targetsequence” to be amplified. Target nucleic acids may be DNA or RNA andmay be either single-stranded or double-stranded. In a preferredembodiment of the invention, the target nucleic acid is DNA. The targetnucleic acid may include other sequences besides the target sequencethat may be amplified. Typical target nucleic acids are or are derivedfrom the Adenovirus genome.

Target sequence. This term refers to the particular nucleotide sequenceof the target nucleic acid that is to be amplified. Where the targetnucleic acid is originally single-stranded, the term “target sequence”will also refer to the sequence complementary to the target sequence aspresent in the target nucleic acid. Where the target nucleic acid isoriginally double-stranded, the term “target sequence” refers to boththe sense (+) and antisense (−) strands. In choosing a target sequence,the skilled artisan will understand that a sequence should be chosen soas to distinguish between unrelated or closely related target nucleicacids. The terms “target(s) a sequence” or “target(s) a target nucleicacid” as used herein in reference to a region of Adenovirus nucleic acidrefers to a process whereby an oligonucleotide stably hybridizes to thetarget sequence in a manner that allows for amplification and/ordetection as described herein. In one embodiment, the oligonucleotide iscomplementary to the targeted Adenovirus nucleic acid sequence andcontains no mismatches. In another embodiment, the oligonucleotide iscomplementary but contains 1; or 2; or 3; or 4; or 5 or more mismatcheswith the targeted Adenovirus nucleic acid sequence. Preferably, theoligonucleotide that stably hybridizes to the Adenovirus nucleic acidsequence includes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 contiguous nucleotidescomplementary to the target sequence. It is understood that at least 10and as many as 50 is an inclusive range such that 10, 50 and each wholenumber there between are included. The term “configured to target asequence” as used herein means that the target hybridizing region of anamplification oligonucleotide is designed to have a polynucleotidesequence that could target a sequence of the referenced Adenovirusregion. Such an amplification oligonucleotide is not limited totargeting that sequence only, but is rather useful as a composition, ina kit or in a method for targeting an Adenovirus target nucleic acid, asis described herein. The term “configured to” denotes an actualarrangement of the polynucleotide sequence configuration of theamplification oligonucleotide target hybridizing sequence.

Isolated. This is meant that a nucleic acid is taken from its naturalmilieu, but the term does not connote any degree of purification.

Fragment. This term as used herein in reference to the Adenovirustargeted nucleic acid sequence refers to a piece of contiguous nucleicacid. In certain embodiments, the fragment includes contiguousnucleotides from Adenovirus target nucleic acid, wherein the number ofcontiguous nucleotides in the fragment are less than that for the entireAdenovirus genome or a gene thereof.

Region. This term refers to a portion of a nucleic acid wherein saidportion is smaller than the entire nucleic acid. For example, when thenucleic acid in reference is an oligonucleotide promoter provider, theterm “region” may be used refer to the smaller promoter portion of theentire oligonucleotide. Similarly, and also as example only, when thenucleic acid is a target nucleic acid, the term “region” may be used torefer to a smaller area of the nucleic acid.

Oligonucleotide. This term may be used interchangeably with “oligomerand “oligo” and refers to a nucleic acid having generally less than1,000 nucleotide (nt) residues, including polymers in a range of fromabout 5 nt residues to about 900 nt residues, from about 10 nt residuesto about 800 nt residues with a lower limit of about 12 to 15 nt and anupper limit of about 40 to 600 nt, and other embodiments are in a rangehaving a lower limit of about 15 to 20 nt and an upper limit of about 22to 100 nt. It is understood that these ranges are exemplary only, and anoligonucleotide may contain each whole number included in the range.Oligonucleotides may be purified from naturally occurring sources, butmay be synthesized using any of a variety of well known enzymatic orchemical methods. The term oligonucleotide does not denote anyparticular function to the reagent; rather, it is used generically tocover all such reagents described herein. An oligonucleotide may servevarious different functions. For example, it may function as a primer ifit is specific for and capable of hybridizing to a complementary strandand can further be extended in the presence of a nucleic acidpolymerase, it may provide a promoter if it contains a sequencerecognized by an RNA polymerase and allows for transcription (eg., a T7provider), and it may function to prevent hybridization or impede primerextension if appropriately situated and/or modified.

As used herein, an oligonucleotide having a nucleic acid sequence“comprising” or “consisting of” or “consisting essentially of” asequence selected from a group of specific sequences means that theoligonucleotide, as a basic and novel characteristic, is capable ofstably hybridizing to a nucleic acid having the exact complement of oneof the listed nucleic acid sequences of the group under stringenthybridization conditions. An exact complement includes the correspondingDNA or RNA sequence.

Corresponds. As used herein, a nucleic acid “corresponds” to a specifiednucleic acid if the nucleic acid is 100% identical or complementary tothe specified nucleic acid.

Substantially corresponding to. As used herein, a nucleic acid“substantially corresponding to” a specified nucleic acid sequence meansthat the referred to oligonucleotide is sufficiently similar to thereference nucleic acid sequence such that the oligonucleotide hassimilar hybridization properties to the reference nucleic acid sequencein that it would hybridize with the same target nucleic acid sequenceunder stringent hybridization conditions. Substantially correspondingnucleic acids vary by at least one nucleotide from the specified nucleicacid. This variation may be stated in terms of a percentage of identityor complementarity between the nucleic acid and the specified nucleicacid. Thus, nucleic acid substantially corresponds to a referencenucleic acid sequence if these percentages of base identity orcomplementarity are from less than 100% to about 80%. In preferredembodiments, the percentage is at least about 85%. In more preferredembodiments, this percentage is at least about 90%; in other preferredembodiments, this percentage is at least about 95%, 96%, 97%, 98% or99%. One skilled in the art will understand that the recited rangesinclude all whole and rational numbers of the range (e.g., 92% or92.377%).

Helper oligonucleotide. A “helper oligonucleotide” or “helper” refers toan oligonucleotide designed to bind to a target nucleic acid and imposea different secondary and/or tertiary structure on the target toincrease the rate and extent of hybridization of a detection probe orother oligonucleotide with the targeted nucleic acid, as described, forexample, in U.S. Pat. No. 5,030,557. Helpers may also be used to assistwith the hybridization to target nucleic acid sequences and function ofprimer, target capture and other oligonucleotides. Helperoligonucleotides may be used in the methods described herein and mayform part of the compositions and kits described herein.

Blocking moiety. As used herein, a “blocking moiety” is a substance usedto “block” the 3′-terminus of an oligonucleotide or other nucleic acidso that it cannot be efficiently extended by a nucleic acid polymerase.

Amplification oligomer. An “amplification oligomer”, which may also becalled an “amplification oligonucleotide” is an oligomer, at least the3′-end of which is complementary to a target nucleic acid (“targethybridizing sequence”), and which hybridizes to a target nucleic acid,or its complement, and participates in a nucleic acid amplificationreaction. An example of an amplification oligomer is a “primer” thathybridizes to a target nucleic acid and contains a 3′ OH end that isextended by a polymerase in an amplification process. Another example ofan amplification oligomer is a “promoter-based amplification oligomer,”which comprises a target hybridizing sequence, and a promoter sequencefor initiating transcription by an appropriate polymerase.Promoter-based amplification oligomers may or may not be extended by apolymerase in a primer-based extension depending upon whether or not the3′ end of the target hybridizing sequence is modified to preventprimer-based extension (e.g., a 3′ blocked end). A promoter-basedamplification oligonucleotide comprising a target hybridizing regionthat is not modified to prevent primer-based extension is referred to asa “promoter-primer.” A promoter-based amplification oligonucleotidecomprising a target hybridizing region that is modified to preventprimer-based extension is referred to as a “promoter-provider.” Sizeranges for amplification oligonucleotides include those comprisingtarget hybridizing regions that are about 10 to about 70 nt long—such asabout 10 to about 60 nt long, about 10 to about 50 nt long, about 10 toabout 40 nt long, about 10 to about 30 nt long or about 10 to about 25nt long or about 15 to 25 nt long. Preferred sizes of amplificationoligomers include those comprising target hybridizing regions that areabout 18, 19, 20, 21, 22 or 23 nt long. An amplification oligomer mayoptionally include modified nucleotides or analogs that are notcomplementary to target nucleic acid in a strict A:T/U, G:C sense. Suchmodified nucleotides or analogs are herein considered mismatched totheir corresponding target sequence.

Oligomers not intended for primer-based extension by a nucleic acidpolymerase may include a blocker group that replaces the 3′OH to preventthe enzyme-mediated extension of the oligomer in an amplificationreaction. For example, blocked amplification oligomers and/or detectionprobes present during amplification may not have functional 3′OH andinstead include one or more blocking groups located at or near the 3′end. In some embodiments a blocking group near the 3′ end and may bewithin five residues of the 3′ end and is sufficiently large to limitbinding of a polymerase to the oligomer. In other embodiments a blockinggroup is covalently attached to the 3′ terminus. Many different chemicalgroups may be used to block the 3′ end, e.g., alkyl groups,non-nucleotide linkers, alkane-diol dideoxynucleotide residues, andcordycepin.

Promoter. This refers to a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

Promoter-provider. As used herein, a “promoter-provider” or “provider”refers to an oligonucleotide comprising first and second regions, andwhich is modified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter—provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The target-hybridizing portion of a promoteroligonucleotide is typically at least 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or 45 nucleotides in length,and may extend up to 50 or more nucleotides in length. The “secondregion” comprises a promoter sequence for an RNA polymerase. Apromoter-provider oligonucleotide is configured so that it is incapableof being extended by an RNA- or DNA-dependent DNA polymerase, (e.g.,reverse transcriptase), preferably by comprising a blocking moiety atits 3′-terminus as described above. This modification differentiatespromoter providers from promoter primers. Preferably, the promoterportion of a promoter primer or provider is a promoter for aDNA-dependent RNA polymerase from E. coli and bacteriophages T7, T3, andSP6, though other promoters or modified version thereof can be used aswell.

Terminating oligonucleotide. As used herein, a “terminatingoligonucleotide” or “blocker oligonucleotide” is an oligonucleotidecomprising a base sequence that is complementary to a region of thetarget nucleic acid in the vicinity of the 5′-end of the targetsequence, so as to “terminate” primer extension of a nascent nucleicacid that includes a priming oligonucleotide, thereby providing adefined 3′-end for the nascent nucleic acid strand.

Amplification. This refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Amplification of “fragments” refers toproduction of an amplified nucleic acid that contains less than thecomplete target nucleic acid or its complement, eg., produced by usingan amplification oligonucleotide that hybridizes to, and initiatespolymerization from, an internal position of the target nucleic acid.Known amplification methods include both thermal and isothermalamplification methods. For some embodiment, isothermal amplificationmethods are preferred. Replicase-mediated amplification, polymerasechain reaction (PCR), ligase chain reaction (LCR), strand-displacementamplification (SDA), and transcription-mediated ortranscription-associated amplification are non-limiting examples ofnucleic acid amplification methods. Replicase-mediated amplificationuses self-replicating RNA molecules, and a replicase such asQB-replicase (eg., U.S. Pat. No. 4,786,600). PCR amplification uses aDNA polymerase, pairs of primers, and thermal cycling to synthesizemultiple copies of two complementary strands of dsDNA or from a cDNA(eg., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCRamplification uses four or more different oligonucleotides to amplify atarget and its complementary strand by using multiple cycles ofhybridization, ligation, and denaturation (eg., U.S. Pat. No. 5,427,930and U.S. Pat. No. 5,516,663). SDA uses a primer that contains arecognition site for a restriction endonuclease and an endonuclease thatnicks one strand of a hemimodified DNA duplex that includes the targetsequence, whereby amplification occurs in a series of primer extensionand strand displacement steps (eg., U.S. Pat. No. 5,422,252; U.S. Pat.No. 5,547,861; and U.S. Pat. No. 5,648,211).

Transcription associated amplification. This method of amplification,also referred to herein as “transcription mediated amplification” (TMA)refers to nucleic acid amplification that uses an RNA polymerase toproduce multiple RNA transcripts from a nucleic acid template. Thesemethods generally employ an RNA polymerase, a DNA polymerase,deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and atemplate complementary oligonucleotide that includes a promotersequence, and optionally may include one or more other oligonucleotides.TMA methods are embodiments of amplification methods used for amplifyingand detecting Adenovirus target sequences as described herein.Variations of transcription associated amplification are well known inthe art as previously disclosed in detail (eg., U.S. Pat. Nos.4,868,105; 5,124,246; 5,130,238; 5,399,491; 5,437,990; 5,554,516; and7,374,885; and PCT Pub. Nos. WO 88/01302; WO 88/10315 and WO 95/03430).The person of ordinary skill in the art will appreciate that thedisclosed compositions may be used in amplification methods based onextension of oligomer sequences by a polymerase.

Real-time amplification. As used herein, the term “real-timeamplification” refers to amplification of target nucleic acid that ismonitored by real-time detection means.

Amplicon. This term, which is used interchangeably with “amplificationproduct”, refers to the nucleic acid molecule generated during anamplification procedure that is complementary or homologous to asequence contained within the target sequence. These terms can be usedto refer to a single strand amplification product, a double strandamplification product or one of the strands of a double strandamplification product.

Probe. A probe, also known as a “detection probe” or “detectionoligonucleotide” are terms referring to a nucleic acid oligomer thathybridizes specifically to a target sequence in a nucleic acid, or in anamplified nucleic acid, under conditions that promote hybridization toallow detection of the target sequence or amplified nucleic acid.Detection may either be direct (e.g., a probe hybridized directly to itstarget sequence) or indirect (e.g., a probe linked to its target via anintermediate molecular structure). Probes may be DNA, RNA, analogsthereof or combinations thereof and they may be labeled or unlabeled. Aprobe's “target sequence” generally refers to a smaller nucleic acidsequence within a larger nucleic acid sequence that hybridizesspecifically to at least a portion of a probe oligomer by standard basepairing. A probe may comprise target-specific sequences and othersequences that contribute to the three-dimensional conformation of theprobe (eg., U.S. Pat. Nos. 5,118,801; 5,312,728; 6,849,412; 6,835,542;6,534,274; and 6,361,945; and US Pub. No. 20060068417). In a preferredembodiment, the detection probe comprises a 2′ methoxy backbone whichcan result in a higher signal being obtained. In another preferredembodiment, the probe comprises a fluorophore covalently attached to the5′-end of the probe and a quencher at the 3′-end. Such probes are knownas Taqman probes.

Stable. By “stable” or “stable for detection” is meant that thetemperature of a reaction mixture is at least 2.deg.C below the meltingtemperature of a nucleic acid duplex.

Label. As used herein, a “label” refers to a moiety or compound joineddirectly or indirectly to a probe that is detected or leads to adetectable signal. Direct labeling can occur through bonds orinteractions that link the label to the probe, including covalent bondsor non-covalent interactions, e.g. hydrogen bonds, hydrophobic and ionicinteractions, or formation of chelates or coordination complexes.Indirect labeling can occur through use of a bridging moiety or “linker”such as a binding pair member, an antibody or additional oligomer, whichis either directly or indirectly labeled, and which may amplify thedetectable signal. Labels include any detectable moiety, such as aradionuclide, ligand (e.g., biotin, avidin), enzyme or enzyme substrate,reactive group, or chromophore (e.g., dye, particle, or bead thatimparts detectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (e.g.,U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels includechemiluminescent compounds, e.g., acridinium ester (“AE”) compounds thatinclude standard AE and derivatives (e.g., U.S. Pat. Nos. 5,656,207,5,658,737, and 5,639,604). Synthesis and methods of attaching labels tonucleic acids and detecting labels are well known (e.g., Sambrook etal., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat.Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). Morethan one label, and more than one type of label, may be present on aparticular probe, or detection may use a mixture of probes in which eachprobe is labeled with a compound that produces a different detectablesignal (e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

Molecular torches. As used herein, structures referred to as “moleculartorches” are designed to include distinct regions ofself-complementarity (“the closing domain”) which are connected by ajoining region (“the target binding domain”) and which hybridize to oneanother under predetermined hybridization assay conditions. All or partof the nucleotide sequences comprising target closing domains may alsofunction as target binding domains. Thus, target closing sequences caninclude, target binding sequences, non-target binding sequences, andcombinations thereof.

Capture oligonucleotide. As used herein, a “capture oligonucleotide,”“target capture oligonucleotide” or “capture probe” refers to a nucleicacid oligomer that specifically hybridizes to a target sequence in atarget nucleic acid by standard base pairing and joins to a bindingpartner on an immobilized probe to capture the target nucleic acid to asupport. One example of a capture oligomer includes an oligonucleotidecomprising two binding regions: a target hybridizing sequence and animmobilized probe-binding region. A variation of this example, the tworegions may be present on two different oligomers joined together by oneor more linkers. Another embodiment of a capture oligomer the targethybridizing sequence is a sequence that includes random or non-randompoly-GU, poly-GT, or poly U sequences to bind non-specifically to atarget nucleic acid and link it to an immobilized probe on a support.(PCT Pub No. WO 2008/016988). The immobilized probe binding region canbe a nucleic acid sequence, referred to as a tail. Tails include asubstantially homopolymeric tail of about 10 to 40 nucleotides (e.g.,A₁₀ to A₄₀), or of about 14 to 33 nt (e.g., T₃A₁₄ to T₃A₃₀), that bindto a complementary immobilized sequence attached to the support particleor support matrix. Thus, a non-limiting example of preferred nucleicacid tails can in some embodiments include T₀₋₄A₁₀₋₃₆ sequences. Anotherexample of a capture oligomer comprises two regions, a targethybridizing sequence and a binding pair member that is not a nucleicacid sequence.

Immobilized oligonucleotide. As used herein, an “immobilizedoligonucleotide”, “immobilized probe” or “immobilized nucleic acid”refers to a nucleic acid binding partner that joins a capture oligomerto a support, directly or indirectly. An immobilized probe joined to asupport facilitates separation of a capture probe bound target fromunbound material in a sample. One embodiment of an immobilized probe isan oligomer joined to a support that facilitates separation of boundtarget sequence from unbound material in a sample. Supports may includeknown materials, such as matrices and particles free in solution, whichmay be made of nitrocellulose, nylon, glass, polyacrylate, mixedpolymers, polystyrene, silane, polypropylene, metal, or othercompositions, of which one embodiment is magnetically attractableparticles. Supports may be monodisperse magnetic spheres (e.g., uniformsize ±5%), to which an immobilized probe is joined directly (viacovalent linkage, chelation, or ionic interaction), or indirectly (viaone or more linkers), where the linkage or interaction between the probeand support is stable during hybridization conditions.

Complementary. By “complementary” is meant that the nucleotide sequencesof similar regions of two single-stranded nucleic acids, or to differentregions of the same single-stranded nucleic acid have a nucleotide basecomposition that allow the single-stranded regions to hybridize togetherin a stable double-stranded hydrogen-bonded region under stringenthybridization or amplification conditions. Sequences that hybridize toeach other may be completely complementary or partially complementary tothe intended target sequence by standard nucleic acid base pairing (e.g.G:C, A:T or A:U pairing). By “sufficiently complementary” is meant acontiguous sequence that is capable of hybridizing to another sequenceby hydrogen bonding between a series of complementary bases, which maybe complementary at each position in the sequence by standard basepairing or may contain one or more residues that are not complementaryby standard A:T/U and G:C pairing, or are modified nucleotides such asabasic residues, modified nucleotides or nucleotide analogs.Sufficiently complementary contiguous sequences typically are at least80%, or at least 90%, complementary to a sequence to which an oligomeris intended to specifically hybridize (a %-complementarity rangeincludes all whole and rational numbers of the range). Sequences thatare “sufficiently complementary” allow stable hybridization of a nucleicacid oligomer with its target sequence under appropriate hybridizationconditions, even if the sequences are not completely complementary. Whena contiguous sequence of nucleotides of one single-stranded region isable to form a series of “canonical” hydrogen-bonded base pairs with ananalogous sequence of nucleotides of the other single-stranded region,such that A is paired with U or T and C is paired with G, thenucleotides sequences are “completely” complementary.

Preferentially hybridize. By “preferentially hybridize” is meant thatunder stringent hybridization assay conditions, an oligonucleotidehybridizes to its target sequences, or replicates thereof, to formstable oligonucleotide: target sequence hybrid, while at the same timeformation of stable oligonucleotide: non-target sequence hybrid isminimized. For example, a probe oligonucleotide preferentiallyhybridizes to a target sequence or replicate thereof to a sufficientlygreater extent than to a non-target sequence, to enable one havingordinary skill in the art to accurately detect the RNA replicates orcomplementary DNA (cDNA) of the target sequence formed during theamplification. Appropriate hybridization conditions are well known inthe art for probe, amplification, target capture, blocker and otheroligonucleotides, may be predicted based on sequence composition, or canbe determined by using routine testing methods (e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, 2^(nd) ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) at §§1.90-1.91,7.37-7.57, 9.47-9.51 and 11.47-11.57, particularly §§9.50-9.51,11.12-11.13, 11.45-11.47 and 11.55-11.57).

Nucleic acid hybrid. By “nucleic acid hybrid” or “hybrid” or “duplex” ismeant a nucleic acid structure containing a double-stranded,hydrogen-bonded region wherein each strand is complementary to theother, and wherein the region is sufficiently stable under stringenthybridization conditions to be detected by means including, but notlimited to, chemiluminescent or fluorescent light detection,autoradiography, or gel electrophoresis. Such hybrids may compriseRNA:RNA, RNA:DNA, or DNA:DNA duplex molecules.

Sample preparation. This refers to any steps or methods that treat asample for subsequent amplification and/or detection of Adenovirusnucleic acids present in the sample. The target nucleic acid may be aminority component in the sample. Sample preparation may include anyknown method of isolating or concentrating components, such as virusesor nucleic acids using standard microbiology methods. Sample preparationmay include physical disruption and/or chemical lysis of cellularcomponents to release intracellular components into a substantiallyaqueous or organic phase and removal of debris, such as by usingfiltration, centrifugation or adsorption. Sample preparation may includeuse of a nucleic acid oligonucleotide that selectively ornon-specifically captures a target nucleic acid and separates it fromother sample components (eg., as described in U.S. Pat. No. 6,110,678and PCT Pub. No. WO 2008/016988).

Separating, purifying. These terms mean that one or more components of asample are removed or separated from other sample components. Samplecomponents include target nucleic acids usually in a generally aqueoussolution phase, which may also include cellular fragments, proteins,carbohydrates, lipids, and other nucleic acids. Separating or purifyingremoves at least 70%, or at least 80%, or at least 95% of the targetnucleic acid from other sample components. Ranges of %-purity includeall whole and rational numbers of the range.

DNA-dependent DNA polymerase. As used herein, a “DNA-dependent DNApolymerase” is an enzyme that synthesizes a complementary DNA copy froma DNA template. Examples are DNA polymerase I from E. coli,bacteriophage T7 DNA polymerase, or DNA polymerases from bacteriophagesT4, Phi-29, M2, or T5. DNA-dependent DNA polymerases may be thenaturally occurring enzymes isolated from bacteria or bacteriophages orexpressed recombinantly, or may be modified or “evolved” forms whichhave been engineered to possess certain desirable characteristics, e.g.,thermostability, or the ability to recognize or synthesize a DNA strandfrom various modified templates. All known DNA-dependent DNA polymerasesrequire a complementary primer to initiate synthesis. It is known thatunder suitable conditions a DNA-dependent DNA polymerase may synthesizea complementary DNA copy from an RNA template. RNA-dependent DNApolymerases typically also have DNA-dependent DNA polymerase activity.

DNA-dependent RNA polymerase. As used herein, a “DNA-dependent RNApolymerase” or “transcriptase” is an enzyme that synthesizes multipleRNA copies from a double-stranded or partially double-stranded DNAmolecule having a promoter sequence that is usually double-stranded. TheRNA molecules (“transcripts”) are synthesized in the 5′-to-3′ directionbeginning at a specific position just downstream of the promoter.Examples of transcriptases are the DNA-dependent RNA polymerase from E.coli and bacteriophages T7, T3, and SP6.

RNA-dependent DNA polymerase. As used herein, an “RNA-dependent DNApolymerase” or “reverse transcriptase” (“RT”) is an enzyme thatsynthesizes a complementary DNA copy from an RNA template. All knownreverse transcriptases also have the ability to make a complementary DNAcopy from a DNA template; thus, they are both RNA- and DNA-dependent DNApolymerases. RTs may also have an RNAse H activity. A primer is requiredto initiate synthesis with both RNA and DNA templates.

Selective RNAse. As used herein, a “selective RNAse” is an enzyme thatdegrades the RNA portion of an RNA:DNA duplex but not single-strandedRNA, double-stranded RNA or DNA. An exemplary selective RNAse is RNAseH. Enzymes possessing the same or similar activity as RNAse H may alsobe used. Selective RNAses may be endonucleases or exonucleases. Mostreverse transcriptase enzymes contain an RNAse H activity in addition totheir polymerase activities. However, other sources of the RNAse H areavailable without an associated polymerase activity. The degradation mayresult in separation of RNA from a RNA:DNA complex. Alternatively, aselective RNAse may simply cut the RNA at various locations such thatportions of the RNA melt off or permit enzymes to unwind portions of theRNA. Other enzymes that selectively degrade RNA target sequences or RNAproducts of the present invention will be readily apparent to those ofordinary skill in the art.

Specificity. The term “specificity,” in the context of an amplificationsystem, is used herein to refer to the characteristic of anamplification system which describes its ability to distinguish betweentarget and non-target sequences dependent on sequence and assayconditions. In terms of nucleic acid amplification, specificitygenerally refers to the ratio of the number of specific ampliconsproduced to the number of side-products (e.g., the signal-to-noiseratio).

Sensitivity. The term “sensitivity” is used herein to refer to theprecision with which a nucleic acid amplification reaction can bedetected or quantitated. The sensitivity of an amplification reaction isgenerally a measure of the smallest copy number of the target nucleicacid that can be reliably detected in the amplification system, and willdepend, for example, on the detection assay being employed, and thespecificity of the amplification reaction, e.g., the ratio of specificamplicons to side-products.

Relative fluorescence unit. As used herein, the term “relativefluorescence unit” (“RFU”) is an arbitrary unit of measurement offluorescence intensity. RFU varies with the characteristics of thedetection means used for the measurement.

Oligonucleotides for the Amplification of Adenovirus

Oligonucleotides for amplifying an Adenovirus target typically compriseat least two amplification oligomers. Some embodiments of the inventionmay utilise, three, four, five, or even six or ten or more amplificationoligomers in, for example, multiplex amplification assays. Thus, by wayof example, oligonucleotides for amplifying the Adenovirus target maycomprise one, two, three, four, or five or more forward amplificationprimers and one, two, three, four, or five or more reverse amplificationprimers. In one embodiment, at least one of the amplification oligomersis configured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 1 to 99 of SEQ IDNo. 47. In another embodiment, at least one of the amplificationoligomers is configured to specifically hybridize to a region within atarget sequence of Adenovirus corresponding to nucleotides 83 to 175 ofSEQ ID No. 47. In one embodiment, at least two amplification oligomersare used, wherein each of said at least two amplification oligomers isfrom 10 to about 50 nucleotides in length and wherein the amplificationoligomers are respectively configured to specifically hybridize toregions within a target sequence of Adenovirus selected from the groupconsisting of from nucleotides 1 to 99 of SEQ ID No. 47 and fromnucleotides 83 to 175 of SEQ ID No. 47 in order to generate an ampliconthat can be subsequently detected. Suitably the amplicon is detectableusing a detection probe. Suitably the amplicon is at least 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 70 or 80 nucleotides in length.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 52 to 99 and/or 40to 87 and/or 1 to 23 and/or 7 to 23 and/or 7 to 45 of SEQ ID No. 47. Inone embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 139 to 155 and/or103 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47. In anotherembodiment, at least two amplification oligomers are used, wherein eachof said at least two amplification oligomers is from 10 to about 50nucleotides in length and wherein the amplification oligomers arerespectively configured to specifically hybridize to regions within atarget sequence of Adenovirus selected from the group consisting of fromnucleotides 52 to 99 and/or 40 to 87 and/or 1 to 23 and/or 7 to 23and/or 7 to 45 of SEQ ID No. 47 and from nucleotides 139 to 155 and/or103 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47 in order togenerate an amplicon that can be subsequently detected.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 52 to 74 and/or 76to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to 18 and/or 7 to 23and/or 28 to 45 and/or 27 to 45 and/or 26 to 45 of SEQ ID No. 47. In oneembodiment, at least one of the amplification oligomers is configured tospecifically hybridize to a region within a target sequence ofAdenovirus corresponding to nucleotides 139 to 155 and/or 103 to 123and/or 159 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47. Inanother embodiment, at least two amplification oligomers are used,wherein each of said at least two amplification oligomers is from 10 toabout 50 nucleotides in length and wherein the amplification oligomersare respectively configured to specifically hybridize to regions withina target sequence of Adenovirus selected from the group consisting offrom nucleotides 52 to 74 and/or 76 to 99 and/or 40 to 56 and/or 65 to87 and/or 1 to 18 and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or26 to 45 of SEQ ID No. 47 and from nucleotides 139 to 155 and/or 103 to123 and/or 159 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47in order to generate an amplicon that can be subsequently detected.

Oligonucleotides for amplifying and/or detecting the Adenovirus targetinclude oligonucleotide sequences selected from the group consisting ofSEQ ID NOS: 1 to 46. Although these sequences are shown as DNAsequences, the sequences include their corresponding RNA sequences, andtheir complementary (eg. completely complementary) DNA or RNA sequences,including the reverse complements thereof.

Target capture oligomers may include a target-specific sequence thatbinds specifically to the Adenovirus target nucleic acid and acovalently linked “tail” sequence (eg. T₀₋₄A₁₀₋₃₆) used in capturing thehybridization complex containing the target nucleic acid to animmobilized sequence on a solid support. Capture oligomers may includeat least one 2′ methoxy linkage. Capture oligomers may include thetarget-specific sequence that binds to Adenovirus nucleic acid attachedto another binding moiety, e.g., a biotinylated sequence that bindsspecifically to immobilized avidin or streptavidin. The tail sequence orbinding moiety binds to an immobilized probe (eg., complementarysequence or avidin) to capture the hybridized target and separate itfrom other sample components by separating the solid support from themixture.

Primer sequences, including promoter primer sequences, bind specificallyto the target nucleic acid or its complementary sequence and may containadditional sequences that are not target-specific, eg., the promotersequence in a promoter primer. A target-specific sequence, with orwithout an attached promoter sequence, may serve as an amplificationoligomer in a variety of in vitro amplification processes. Embodimentsof the Adenovirus assays may use amplification methods that requiremultiple cycling reaction temperatures, such as PCR (U.S. Pat. Nos.4,683,195, 4,683,202, and 4,800,159), or may be substantially isothermalas in, for example, transcription associated amplification methods, suchas TMA or NASBA (e.g., U.S. Pat. Nos. 5,399,491, 5,480,784, 5,824,518,5,888,779, 5,786,183, 5,437,990, 5,130,238, 4,868,105, and 5,124,246,and PCT Nos. WO 8801302 and WO 8810315). The Adenovirus assays may useamplification systems that are detected during the amplification process(e.g., real time detection) by including probes that emitdistinguishable fluorescent signals when the probe is bound to theintended target sequence made during the amplification process. Probesfor real time detection include those referred to as “molecular beacon”or “molecular switch” probes (e.g., U.S. Pat. Nos. 5,118,801 and5,312,728, Lizardi et al., U.S. Pat. Nos. 5,925,517 and 6,150,097, Tyagiet al., Giesendorf et al., 1998, Clin. Chem. 44(3):482-6) and “moleculartorch” probes (e.g., U.S. Pat. Nos. 6,835,542 and 6,849,412, Becker etal.). Generally, such probes include a reporter dye attached to one endof the probe oligomer (e.g., FAM™, TET™, JOE™, VIC™) and a quenchercompound (e.g., TAMRA™, BHQ1 or non-fluorescent quencher) attached tothe other end of the probe oligomer, and signal production depends onwhether the two ends with their attached compounds are in closeproximity or separated.

The assay to detect Adenovirus in a sample includes the steps ofamplifying a target region in the target Adenovirus nucleic acidcontained in a sample by using amplification oligomers or primersspecific for the intended target region, and detecting the amplifiednucleic acid by hybridizing it to a probe sequence. Preferred assays usea PCR or transcription-associated amplification reaction and detectionis at the end of the amplification reaction. For detection, theamplified nucleic acid may be labeled and bound to an unlabeled probe,but preferred embodiments bind a labeled probe to the amplified nucleicacid. For real-time detection, a labeled probe may be used that isdetected in a homogeneous system.

Embodiments of amplification oligomers specific for Adenovirus nucleicacid include the amplification oligomers comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NOS: 1 to 9, 11 to 16, 25 to 28, 31 to35, 38, and 42 to 46 or a combination of two or more thereof. Accordingto one embodiment, at least one first amplification oligomer comprises,consists of consists essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NOS: 1, 5, 11, 12, 25, 26,31, 32, 33, 34, 35 or 38 or a combination of two or more thereof.According to one embodiment, at least one second amplification oligomercomprises, consists of consists essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NOS: 2, 3, 6, 7,8, 9, 13, 14, 15, 16, 27, 28, 42, 43, 44, 45 or 46 or a combination oftwo or more thereof.

Combinations of amplification oligomers specific for Adenovirus nucleicacid are therefore contemplated.

According to one embodiment, at least one first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 1 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence as set forth inSEQ ID NO: 2 or SEQ ID NO: 3.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 5 is used in combinationwith at least one second amplification oligomer comprising, consistingor consisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQID NO: 9 or a combination of two or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 11 and/or SEQ ID NO: 12is used in combination with at least one second amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO: 15 and SEQ ID NO: 16 or a combination of two or morethereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 25 and/or SEQ ID NO: 26is used in combination with at least one second amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 27 and/or SEQ ID NO: 28. Accordingto one embodiment, at least two first amplification oligomerscomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 25 and SEQ ID NO: 26 are used incombination with at least two second amplification oligomers comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO: 27 and SEQ ID NO: 28.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 and SEQID NO: 38 or a combination of two or more thereof is used in combinationwith at least one second amplification oligomer comprising, consistingor consisting essentially of a target hybridizing sequence as set forthin SEQ ID NO: 27 and/or SEQ ID NO: 28.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 1 is used in combinationwith at least one second amplification oligomer comprising, consistingor consisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 or SEQ ID NO:28 or a combination of two or morethereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 5 is used in combinationwith at least one second amplification oligomer comprising, consistingor consisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 or SEQ ID NO:28 or a combination of two or morethereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 5 is used in combinationwith at least one second amplification oligomer comprising, consistingor consisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 11 is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination oftwo or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 12 is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination oftwo or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 25 is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination oftwo or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 26 is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination oftwo or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 31 is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination oftwo or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 32 is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination oftwo or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:2.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:3.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:6.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:7.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:8.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:9.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:13.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:14.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:15.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11 SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQID NO:38 or a combination of two or more thereof is used in combinationwith at least one second amplification oligomer comprising, consistingor consisting essentially of a target hybridizing sequence as set forthin SEQ ID NO:16.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:27.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence selected from the group consisting of SEQ ID NO: 1,SEQ ID NO: 5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 andSEQ ID NO:38 or a combination of two or more thereof is used incombination with at least one second amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO:28.

The methods for detecting Adenovirus nucleic acid optionally include adetecting step that uses at least one probe that binds specifically tothe amplified Adenovirus product (RNA or DNA amplicon, preferably DNAamplicon). Preferably, the probe is labeled and produces a signaldetected in a homogeneous system, that is, without separation of boundprobe from unbound probe. Other examples of probes may be labeled with afluorescent compound which emits a detectable signal only when the probeis bound to its target, e.g., molecular switch, beacon, Taqman or torchprobes as further described herein.

Probes comprise polynucleotides or polynucleotide analogs and optionallymay carry a detectable label covalently bonded thereto. Nucleosides ornucleoside analogs of the probe may comprise nitrogenous heterocyclicbases, or base analogs, where the nucleosides are linked together, forexample by phospohdiester bonds to form a polynucleotide. Accordingly, aprobe may comprise conventional ribonucleic acid (RNA) and/ordeoxyribonucleic acid (DNA), but also may comprise chemical analogs ofthese molecules. The “backbone” of a probe may be made up of a varietyof linkages known in the art, including one or more sugar-phosphodiesterlinkages, peptide-nucleic acid bonds (sometimes referred to as “peptidenucleic acids” as described by Hyldig-Nielsen et al., PCT Intl Pub. No.WO 95/32305), phosphorothioate linkages, methylphosphonate linkages orcombinations thereof. Sugar moieties of the probe may be either riboseor deoxyribose, or similar compounds having known substitutions, suchas, for example, 2′-O-methyl ribose and 2′ halide substitutions (e.g.,2′-F). The nitrogenous bases may be conventional bases (A, G, C, T, U),known analogs thereof (e.g., inosine or “I”; see The Biochemistry of theNucleic Acids 5-36, Adams et al., ed., 11^(th) ed., 1992), knownderivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines and deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or a replacement substituent at the 2, 6or 8 positions, 2-amino-6-methylaminopurine, O⁶-methylguanine,4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines (see, Cook,PCT Intl Pub. No. WO 93/13121) and “abasic” residues where the backboneincludes no nitrogenous base for one or more residues of the polymer(see Arnold et al., U.S. Pat. No. 5,585,481). A probe may comprise onlyconventional sugars, bases and linkages found in RNA and DNA, or mayinclude both conventional components and substitutions (e.g.,conventional bases linked via a methoxy backbone, or a nucleic acidincluding conventional bases and one or more base analogs). Whileoligonucleotide probes of different lengths and base composition may beused for detecting Adenovirus nucleic acids, preferred probes in thisinvention have lengths of up to 100 nucleotides, and more preferablyhave lengths of up to 60 nucleotides. Preferred length ranges for theinvented oligonucleotides are from 10 to 100 bases in length, or morepreferably between 15 and 50 bases in length, or still more preferablybetween 15 and 40 bases in length, or still more preferably between 15and 30 bases in length. However, the specific probe sequences describedherein also may be provided in a nucleic acid cloning vector ortranscript or other longer nucleic acid and still can be used fordetecting Adenovirus nucleic acids.

In one embodiment, one or more detection probes are configured to detecta sequence in a region corresponding to nucleotides 74 to 139 of SEQ IDNO:47; and/or nucleotides 56 to 103 of SEQ ID NO:47; and/or nucleotides18 to 83 of SEQ ID NO:47; and/or nucleotides 23 to 83 of SEQ ID NO:47;and/or nucleotides 23 to 83 of SEQ ID NO:47; and/or nucleotides 23 to 83of SEQ ID NO:47 and/or nucleotides 52 to 99 of SEQ ID NO:47.

In another embodiment, one or more of the detection probes areconfigured to target a sequence in a region corresponding to nucleotides76 to 99 and/or nucleotides 65 to 87 and/or nucleotides 53 to 76 and/ornucleotides 53 and/or 74 or nucleotides 53 and/or 72 or nucleotides 52to 71.

Probes for the specific detection of Adenovirus sequences includeoligomers selected from the group consisting of SEQ ID Nos. 4, 10, 17,18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or a combinationof two or more thereof.

Although these sequences are shown as DNA sequences, the sequencesinclude their corresponding RNA sequences, and their complementary (eg.completely complementary) DNA or RNA sequences, including the reversecomplements thereof.

Assays for detection of Adenovirus nucleic acid may include an internalcontrol (IC) nucleic acid that is amplified and detected by usingIC-specific primers and probe in the same reaction mixtures used forAdenovirus nucleic acid amplification and detection. Amplification anddetection of the IC-specific sequence demonstrates that assay reagentsand conditions were properly used even when no Adenovirus-specificsignal is detected for a tested sample (i.e., negative samples). The ICmay be used as an internal calibrator for the assay that provides aquantitative result. The IC may be a randomized sequence derived from anaturally occurring source that is not Adenovirus.

Combinations and Compositions of Oligonucleotides for the Amplificationand Detection of Adenovirus

Combinations of oligomers and probes that can be used for theamplification and detection of Adenovirus are also disclosed.

Oligonucleotides for amplifying and detecting the Adenovirus targettypically comprise at least two amplification oligomers and at least oneprobe. Some embodiments of the invention may utilise, three, four, five,or even six or more amplification oligomers and two, three, four, fiveor even six or more probes. Thus, by way of example, oligonucleotidesfor amplifying and detecting the Adenovirus target may comprise one, twoor three or more forward amplification primers together with one, two orthree or more reverse amplification primers together with one, two,three, four, five or even six or more probes.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 1 to 99 of SEQ IDNo. 47 and a probe is configured to detect a sequence in a regioncorresponding to nucleotides 52 to 99 of SEQ ID NO:47.

In another embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 83 to 175 of SEQ IDNo. 47 and a probe is configured to detect a sequence in a regioncorresponding to nucleotides 52 to 99 of SEQ ID NO:47.

In one embodiment, at least two amplification oligomers are used,wherein each of said at least two amplification oligomers is from 10 toabout 50 nucleotides in length and wherein the amplification oligomersare respectively configured to specifically hybridize to regions withina target sequence of Adenovirus selected from the group consisting offrom nucleotides 1 to 99 of SEQ ID No. 47 and from nucleotides 83 to 175of SEQ ID No. 47 and a probe is configured to detect a sequence in aregion corresponding to nucleotides 52 to 99 of SEQ ID NO:47.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 52 to 99 and/or 40to 87 and/or 1 to 23 and/or 7 to 23 and/or 7 to 45 of SEQ ID No. 47 anda probe is configured to detect a sequence in a region corresponding tonucleotides 52 to 99 of SEQ ID NO:47—such as nucleotides 76 to 99 ornucleotides 65 to 87 or nucleotides 53 to 76 or nucleotides 53 to 74 ornucleotides 53 to 72 or nucleotides 52 to 71.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 139 to 155 and/or103 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47 and a probeis configured to detect a sequence in a region corresponding tonucleotides 52 to 99 of SEQ ID NO:47—such as nucleotides 76 to 99 ornucleotides 65 to 87 or nucleotides 53 to 76 or nucleotides 53 to 74 ornucleotides 53 to 72 or nucleotides 52 to 71.

In another embodiment, at least two amplification oligomers are used,wherein each of said at least two amplification oligomers is from 10 toabout 50 nucleotides in length and wherein the amplification oligomersare respectively configured to specifically hybridize to regions withina target sequence of Adenovirus selected from the group consisting offrom nucleotides 52 to 99 and/or 40 to 87 and/or 1 to 23 and/or 7 to 23and/or 7 to 45 of SEQ ID No. 47 and from nucleotides 139 to 155 and/or103 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47 and a probeis configured to detect a sequence in a region corresponding tonucleotides 52 to 99 of SEQ ID NO:47—such as nucleotides 76 to 99 ornucleotides 65 to 87 or nucleotides 53 to 76 or nucleotides 53 to 74 ornucleotides 53 to 72 or nucleotides 52 to 71.

In one embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 52 to 74 and/or 76to 99 and/or 40 to 56 and/or 65 to 87 and/or 1 to 18 and/or 7 to 23and/or 28 to 45 and/or 27 to 45 and/or 26 to 45 of SEQ ID No. 47 and aprobe is configured to detect a sequence in a region corresponding tonucleotides 52 to 99 of SEQ ID NO:47—such as nucleotides 76 to 99 ornucleotides 65 to 87 or nucleotides 53 to 76 or nucleotides 53 to 74 ornucleotides 53 to 72 or nucleotides 52 to 71.

In another embodiment, at least one of the amplification oligomers isconfigured to specifically hybridize to a region within a targetsequence of Adenovirus corresponding to nucleotides 139 to 155 and/or103 to 123 and/or 159 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ IDNo. 47 and a probe is configured to detect a sequence in a regioncorresponding to nucleotides 52 to 99 of SEQ ID NO:47—such asnucleotides 76 to 99 or nucleotides 65 to 87 or nucleotides 53 to 76 ornucleotides 53 to 74 or nucleotides 53 to 72 or nucleotides 52 to 71.

In another embodiment, at least two amplification oligomers are used,wherein each of said at least two amplification oligomers is from 10 toabout 50 nucleotides in length and wherein the amplification oligomersare respectively configured to specifically hybridize to regions withina target sequence of Adenovirus selected from the group consisting offrom nucleotides 52 to 74 and/or 76 to 99 and/or 40 to 56 and/or 65 to87 and/or 1 to 18 and/or 7 to 23 and/or 28 to 45 and/or 27 to 45 and/or26 to 45 of SEQ ID No. 47 and from nucleotides 139 to 155 and/or 103 to123 and/or 159 to 175 and/or 83 to 99 and/or 83 to 98 of SEQ ID No. 47and a probe is configured to detect a sequence in a region correspondingto nucleotides 52 to 99 of SEQ ID NO:47—such as nucleotides 76 to 99 ornucleotides 65 to 87 or nucleotides 53 to 76 or nucleotides 53 to 74 ornucleotides 53 to 72 or nucleotides 52 to 71.

According to one embodiment, a first amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO: 1 is used in combination with a secondamplification oligomer comprising, consisting or consisting essentiallyof a target hybridizing sequence as set forth in SEQ ID NO: 2 and/or SEQID NO: 3 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 5 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence as set forth inSEQ ID NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8 and/or SEQ ID NO: 9together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 10.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 11 and/or SEQ ID NO: 12is used in combination with at least one second amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 13 and/or SEQ ID NO: 14 and/or SEQID NO: 15 and/or SEQ ID NO: 16 together with a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24,29, 30, 36, 37, 39 and 40 or a combination of two or more thereof,preferably, a probe comprising, consisting or consisting essentially ofa sequence selected from the group consisting of SEQ ID Nos 17 to 24 ora combination of two or more thereof.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 25 and/or SEQ ID NO: 26is used in combination with at least one second amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 27 and/or SEQ ID NO: 28 togetherwith a probe comprising, consisting or consisting essentially of asequence selected from the group consisting of SEQ ID Nos. 4, 10, 17,18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or a combinationof two or more thereof, preferably, SEQ ID Nos 29 and/or 30.

According to another embodiment, at least one first amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 31 and/or SEQ ID NO: 32and/or SEQ ID NO: 33 and/or SEQ ID NO: 34 and/or SEQ ID NO: 35 and/orSEQ ID NO: 38 is used in combination with at least one secondamplification oligomer comprising, consisting or consisting essentiallyof a target hybridizing sequence as set forth in SEQ ID NO: 27 and/orSEQ ID NO: 28 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 36, 37, 39 and 40 or a combination of twoor more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 1 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence as set forth inSEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8, SEQ ID NO: 9, SEQID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:27 and SEQID NO:28 or a combination of two or more thereof together with a probecomprising, consisting or consisting essentially of a sequence selectedfrom the group consisting of SEQ ID Nos. 4, 10, 17, 18, 19, 20, 21, 22,23, 24, 29, 30, 36, 37, 39 and 40 or a combination of two or morethereof, preferably, SEQ ID No. 4

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 5 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, SEQ ID No. 10.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 11 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 17 to 24 or a combination of two or morethereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 12 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 17 to 24 or a combination of two or morethereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 25 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 29 and 30 or a combination of two or morethereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 26 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 29 and 30 or a combination thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 31 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 36, 37, 39 and 40 or a combination of twoor more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 32 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 36, 37, 39 and 40 or a combination of twoor more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 33 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO: 9, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:16, SEQ ID NO:27 and SEQ ID NO:28 or a combination of two or morethereof together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, a probe comprising,consisting or consisting essentially of a sequence selected from thegroup consisting of SEQ ID Nos 36, 37, 39 and 40 or a combination of twoor more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:2together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:3together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:6together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:7together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:8together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:9together with a probe comprising, consisting or consisting essentiallyof a sequence selected from the group consisting of SEQ ID Nos. 4, 10,17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 or acombination of two or more thereof, preferably, SEQ ID No. 4.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ IDNO:13 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, with a probecomprising, consisting or consisting essentially of a sequence selectedfrom the group consisting of SEQ ID Nos 17 to 24 or a combinationthereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ IDNO:14 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, with a probecomprising, consisting or consisting essentially of a sequence selectedfrom the group consisting of SEQ ID Nos 17 to 24 or or a combination oftwo or more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ IDNO:15 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, with a probecomprising, consisting or consisting essentially of a sequence selectedfrom the group consisting of SEQ ID Nos 17 to 24 or a combination of twoor more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ IDNO:16 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, with a probecomprising, consisting or consisting essentially of a sequence selectedfrom the group consisting of SEQ ID Nos 17 to 24 or a combination of twoor more thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ IDNO:27. together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, with a probecomprising, consisting or consisting essentially of SEQ ID No 29 or 30or a combination thereof.

According to another embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:31,SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:38or a combination of two or more thereof is used in combination with asecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ IDNO:28 together with a probe comprising, consisting or consistingessentially of a sequence selected from the group consisting of SEQ IDNos. 4, 10, 17, 18, 19, 20, 21, 22, 23, 24, 29, 30, 36, 37, 39 and 40 ora combination of two or more thereof, preferably, with a probecomprising, consisting or consisting essentially of SEQ ID No 29 or 30or a combination thereof.

Preferred Combinations and Compositions for the Amplification and/orDetection of Adenovirus

In one preferred embodiment, a first amplification oligomer comprising,consisting or consisting essentially of a target hybridizing sequence asset forth in SEQ ID NO: 5 is used in combination with at least onesecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence selected from the groupconsisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO:8 and SEQ ID NO: 9or a combination of two or more thereof. According to a furtherpreferred embodiment, any of these combinations are used together withat least one probe comprising, consisting or consisting essentially ofthe sequence set forth in SEQ ID No. 10.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 11 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence selected fromthe group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 andSEQ ID NO: 16 or a combination of two or more thereof. According to afurther preferred embodiment, any of these combinations are usedtogether with at least one probe comprising, consisting or consistingessentially of the sequence set forth in SEQ ID No. 19 or SEQ ID No. 20or a combination thereof.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 12 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence a selected fromthe group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 andSEQ ID NO: 16 or a combination of two or more thereof. According to afurther preferred embodiment, any of these combinations are usedtogether with at least one probe comprising, consisting or consistingessentially of the sequence set forth in SEQ ID No. 19 or SEQ ID No. 20or a combination thereof.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 12 is used in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of a target hybridizing sequence as set forth inSEQ ID NO: 15 or SEQ ID NO: 16 or a combination thereof. According to afurther preferred embodiment, this combination is used together with atleast one probe comprising, consisting or consisting essentially of asequence selected from the group consisting of SEQ ID No. 21, SEQ ID No.22, SEQ ID No. 23 and SEQ ID No. 24 or a combination of two or morethereof.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 25 or SEQ ID NO: 26 or a combinationthereof is used in combination with at least one second amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 27 or SEQ ID NO: 28 or acombination thereof (eg. SEQ ID Nos. 25, 26, 27 and 28; SEQ ID Nos. 26,27 and 28; SEQ ID Nos. 25, 27 and 28; SEQ ID Nos. 25, 26 and 28; SEQ IDNos. 25, 26 and 27). According to a further preferred embodiment, anyone or more of these combinations can be used together with at least oneprobe comprising, consisting or consisting essentially of the sequenceselected from the group consisting of SEQ ID No. 21, SEQ ID No. 22, SEQID No. 23, SEQ ID No. 24, SEQ ID No. 29 and SEQ ID No. 30 or acombination of two or more thereof.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 31 or SEQ ID NO: 26 or a combinationthereof is used in combination with at least one second amplificationoligomer comprising, consisting or consisting essentially of a targethybridizing sequence as set forth in SEQ ID NO: 27 or SEQ ID NO: 28 or acombination thereof. According to a further preferred embodiment, anyone or more of these combinations can be used together with at least oneprobe comprising, consisting or consisting essentially of the sequenceselected from the group consisting of SEQ ID No. 21, SEQ ID No. 22, SEQID No. 23 and SEQ ID No. 24 or a combination of two or more thereof.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence selected from the group consisting of SEQ ID NO: 33, SEQ ID NO:34 and SEQ ID NO: 35 or a combination of two or more thereof (eg. SEQ IDNos. 33 and 35 or 34 and 35) is used in combination with at least onesecond amplification oligomer comprising, consisting or consistingessentially of a target hybridizing sequence as set forth in SEQ ID NO:27 or SEQ ID NO: 28 or a combination thereof. According to a furtherpreferred embodiment, any one or more of these combinations can be usedtogether with at least one probe comprising, consisting or consistingessentially of the sequence selected from the group consisting of SEQ IDNo. 21, SEQ ID No. 22, SEQ ID No. 23 and SEQ ID No. 24 or a combinationof two or more thereof.

In another preferred embodiment, a first amplification oligomercomprising, consisting or consisting essentially of a target hybridizingsequence as set forth in SEQ ID NO: 25 or SEQ ID NO: 26 or a combinationof two or more thereof is used in combination with at least one secondamplification oligomer comprising, consisting or consisting essentiallyof SEQ ID NO: 27 or SEQ ID NO: 28 or a combination thereof (eg. SEQ IDNos 25, 26, 27 and 28; SEQ ID Nos 26, 27 and 28; SEQ ID Nos 25, 26 and28; SEQ ID Nos 25, 26 and 27; SEQ ID Nos 25, 27 and 28; SEQ ID Nos 25and 27; SEQ ID Nos 25 and 28; SEQ ID Nos 26 and 27 and SEQ ID Nos 26 and28). According to a further preferred embodiment, any one or more ofthese combinations can be used together with at least one probecomprising, consisting or consisting essentially of the sequence setforth in SEQ ID No. 36 or SEQ ID No. 37 or a combination thereof. Thus,for example, a particular preferred combination is any of SEQ ID Nos 25,26, 27 and 28; SEQ ID Nos 26, 27 and 28; SEQ ID Nos 25, 26 and 28; SEQID Nos 25, 26 and 27; SEQ ID Nos 25, 27 and 28; SEQ ID Nos 25 and 27;SEQ ID Nos 25 and 28; SEQ ID Nos 26 and 27 and SEQ ID Nos 26 and 28 incombination with SEQ ID No. 36 and/or SEQ ID No. 37.

Sample Preparation

Preparation of samples for amplification and detection of Adenovirussequences may include methods of separating and/or concentrating virusescontained in a sample from other sample components. Sample preparationmay include routine methods of disrupting samples or lysing samples torelease intracellular contents, including Adenovirus nucleic acids orgenetic sequences comprising Adenovirus nucleic acid. Sample preparationbefore amplification may include an optional step of target capture tospecifically or non-specifically separate the target nucleic acids fromother sample components. Nonspecific target capture methods may involveselective precipitation of nucleic acids from a substantially aqueousmixture, adherence of nucleic acids to a support that is washed toremove other sample components, other methods of physically separatingnucleic acids from a mixture that contains Adenovirus nucleic acid andother sample components.

Amplification of the Adenovirus Target Region

Amplifying the Adenovirus target region using two or more primers may beaccomplished using a variety of known nucleic acid amplificationreactions. For example, amplification may be achieved using PCRamplification (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159,Mullis et al.) to produce multiple DNA strands by using thermocyclingreactions that separate dsDNA and primers specific for portions of theseparated strands to make additional dsDNA molecules by using a DNApolymerase. Well known variations of the basic PCR method may also beused, e.g., PCR coupled with real-time detection—such as Taqman PCR. Onedisadvantage of PCR is the need of a thermocycler to heat and cool theamplification mixture to denature the DNA. As well as PCR, a variety ofother techniques have been developed for detection and amplification ofspecific sequences. One example is the ligase chain reaction (LCR). Inaddition to conventional methods of DNA amplification that rely on thethermal denaturation of the target during the amplification reaction, anumber of methods have been described that do not require templatedenaturation during the amplification reaction and are thus termedisothermal amplification technologies. Examples of isothermalamplification are Strand Displacement Amplification (SDA), TranscriptionMediated Amplification (TMA) and Nucleic Acid Sequence BasedAmplification (NASBA) that use an RNA polymerase to copy RNA sequencesbut not corresponding genomic DNA. Other DNA-based isothermal techniquesinclude Rolling Circle Amplification (RCA), Ramification Amplification(RAM) and Helicase-Dependent isothermal DNA amplification (HDA).

In one embodiment, the amplification method is TMA. A TMA-based assayproduces many RNA transcripts (amplicons) from a single copy of targetnucleic acid and the amplicons are detected to indicate the presence ofthe target Adenovirus in the sample. Briefly, in TMA-based assays, apromoter-primer hybridizes specifically to the target sequence andreverse transcriptase (RT) that includes RNaseH activity creates a firststrand cDNA by extension from the 3′ end of the promoter-primer anddigests the template strand. The cDNA is then bound by a second primerand a new strand of DNA is synthesized from the end of the second primerusing RT to create a double-stranded DNA (dsDNA) containing a functionalpromoter sequence. RNA polymerase specific for that promoter binds tothe promoter sequence and multiple RNA transcripts are produced, whicheach can act as a template for additional sequence replication using thesame steps used for the initial template. Thus, large amounts ofsingle-stranded amplified product are made using substantiallyisothermal reaction conditions.

Amplification methods that use TMA amplification may include thefollowing steps. Briefly, a target nucleic acid containing the targetsequence to be amplified is provided. A first amplification oligomer isbrought in contact with that target nucleic acid by hybridizing to thetarget sequence. The first amplification oligomer may be a primer or apromoter primer. A suitable nucleic acid polymerase then generates anucleic acid strand amplification product that is complementary to thetarget nucleic acid target sequence. Using a primer as the firstamplification oligomer, then the second amplification oligomer is apromoter primer or promoter provider. A suitable nucleic acid polymeraseuses the newly generated amplification product to which thepromoter-based oligomer is hybridized as a primer to make acomplementary strand of the unhybridized promoter sequence. If thesecond amplification oligomer is a promoter primer, then a complementarycopy of the amplification product hybridized by the second amplificationoligomer is also generated. The now double stranded promoter sequence ofthe promoter-based amplification is used by a suitable RNA polymerase toinitiate transcription and make RNA transcript amplification products.The first amplification oligomer primer can then hybridize thetranscribed amplification products and the steps can repeat. When thetarget nucleic acid is DNA the first amplification oligomer is apromoter primer and the second amplification is a primer. Amplificationgenerally proceeds as described above, and as is described in the art.See e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491;5,437,990; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302; WO88/10315 and WO 95/03430 describing TMA and other variations oftranscription-associated amplification. The amplified products may bedetected in real-time during amplification, or at the end of theamplification reaction. Detection may be performed by a number ofmethods. Probe-based detection methods use an oligonucleotide probecomprising a target hybridizing sequence that binds specifically to atarget sequence contained in the amplification products. Detection of asignal resulting from the bound probes indicates the presence of thetarget nucleic acid in the sample.

Nucleic Acid Detection

Detection of the nucleic acids may be accomplished by a variety ofmethods. Detection methods may use nucleic acid probes comprising atarget hybridizing sequence that is complementary to a portion of theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481). Directly or indirectly labeledprobes that specifically associate with the amplified product provide adetectable signal that indicates the presence of the target nucleic acidin the sample. For example, if the target nucleic acid is AdenovirusDNA, the amplified product will contain a sequence in or complementaryto an Adenovirus target sequence. A probe is configured to bind directlyor indirectly to a portion of the amplification product to indicate thepresence of Adenovirus in the tested sample.

Amplified products may be detected in real-time during amplification, orat the end of the amplification reaction. Detection may be performed bya number of methods. Probe-based detection methods use anoligonucleotide probe comprising a target hybridizing sequence thatbinds specifically to a target sequence contained in the amplificationproducts. Detection of a signal resulting from the bound probesindicates the presence of the target nucleic acid in the sample.

Essentially any labeling and detection system that can be used formonitoring specific nucleic acid hybridization can be used inconjunction with the present invention. Included among the collection ofuseful labels are radiolabels, enzymes, haptens, linkedoligonucleotides, chemi luminescent molecules, fluorescent moieties(either alone or in combination with “quencher” moieties), andredox-active moieties that are amenable to electronic detection methods.Preferred chemiluminescent molecules include acridinium esters of thetype disclosed by Arnold et al., in U.S. Pat. No. 5,283,174 for use inconnection with homogenous protection assays, and of the type disclosedby Woodhead et al., in U.S. Pat. No. 5,656,207 for use in connectionwith assays that quantify multiple targets in a single reaction.Electronic labeling and detection approaches are disclosed in U.S. Pat.Nos. 5,591,578 and 5,770,369, and the published international patentapplication WO 98/57158.

Redox active moieties useful as labels include transition metals such asCd, Mg, Cu, Co, Pd, Zn, Fe and Ru. Particularly preferred detectablelabels for probes in accordance with the present invention aredetectable in homogeneous assay systems (i.e., where, in a mixture,bound labeled probe exhibits a detectable change, such as stability ordifferential degradation, compared to unbound labeled probe). Whileother homogeneously detectable labels, such as fluorescent labels andelectronically detectable labels, are intended for use in the practiceof the present invention, a preferred label for use in homogenous assaysis a chemiluminescent compound (e.g., as described by Woodhead et al.,in U.S. Pat. No. 5,656,207; by Nelson et al., in U.S. Pat. No.5,658,737; or by Arnold et al., in U.S. Pat. No. 5,639,604).Chemiluminescent labels may include acridinium ester (“AE”) compounds,such as standard AE or derivatives thereof, such as naphthyl-AE,ortho-AE, 1- or 3-methyl-AE, 2,7-dimethyl-AE, 4,5-dimethyl-AE,ortho-dibromo-AE, ortho-dimethyl-AE, meta-dimethyl-AE, ortho-methoxy-AE,ortho-methoxy(cinnamyl)-AE, ortho-methyl-AE, ortho-fluoro-AE, 1- or3-methyl-ortho-fluoro-AE, 1- or 3-methyl-meta-difluoro-AE, and2-methyl-AE.

Another example of a hybridization assay probe that may be used inconjunction with the invention is a structure commonly referred to as a“Molecular Beacon.” Molecular Beacons comprise nucleic acid moleculeshaving a target complementary sequence, an affinity pair (or nucleicacid arms) holding the probe in a closed conformation in the absence ofa target nucleic acid sequence, and a label pair that interacts when theprobe is in a closed conformation. Hybridization of the target nucleicacid and the target complementary sequence separates the members of theaffinity pair, thereby shifting the probe to an open conformation. Theshift to the open conformation is detectable due to reduced interactionof the label pair, which may be, for example, a fluorophore and aquencher (e.g., DABCYL and EDANS). Molecular Beacons are fully describedin U.S. Pat. No. 5,925,517. Molecular beacons useful for detectingAdenovirus-specific nucleic acid sequences may be created by appendingto either end of one of the probe sequences disclosed herein, a firstnucleic acid arm comprising a fluorophore and a second nucleic acid armcomprising a quencher moiety. In this configuration, theAdenovirus-specific probe sequence disclosed herein serves as thetarget-complementary “loop” portion of the resulting molecular beacon.

Molecular beacons preferably are labeled with an interactive pair ofdetectable labels. Examples of detectable labels that are preferred asmembers of an interactive pair of labels interact with each other byFRET or non-FRET energy transfer mechanisms. Fluorescence resonanceenergy transfer (FRET) involves the radiationless transmission of energyquanta from the site of absorption to the site of its utilization in themolecule, or system of molecules, by resonance interaction betweenchromophores, over distances considerably greater than interatomicdistances, without conversion to thermal energy, and without the donorand acceptor coming into kinetic collision. The “donor” is the moietythat initially absorbs the energy, and the “acceptor” is the moiety towhich the energy is subsequently transferred, hi addition to FRET, thereare at least three other “non-FRET” energy transfer processes by whichexcitation energy can be transferred from a donor to an acceptormolecule.

When two labels are held sufficiently close that energy emitted by onelabel can be received or absorbed by the second label, whether by a FRETor non-FRET mechanism, the two labels are said to be in “energy transferrelationship” with each other. This is the case, for example, when amolecular beacon is maintained in the closed state by formation of astem duplex, and fluorescent emission from a fluorophore attached to onearm of the probe is quenched by a quencher moiety on the opposite arm.

Highly preferred label moieties for the molecular beacons include afluorophore and a second moiety having fluorescence quenching properties(i.e., a “quencher”). In this embodiment, the characteristic signal islikely fluorescence of a particular wavelength, but alternatively couldbe a visible light signal. When fluorescence is involved, changes inemission are preferably due to FRET, or to radiative energy transfer ornon-FRET modes. When a molecular beacon having a pair of interactivelabels in the closed state is stimulated by an appropriate frequency oflight, a fluorescent signal is generated at a first level, which may bevery low. When this same probe is in the open state and is stimulated byan appropriate frequency of light, the fluorophore and the quenchermoieties are sufficiently separated from each other that energy transferbetween them is substantially precluded. Under that condition, thequencher moiety is unable to quench the fluorescence from thefluorophore moiety. If the fluorophore is stimulated by light energy ofan appropriate wavelength, a fluorescent signal of a second level,higher than the first level, will be generated. The difference betweenthe two levels of fluorescence is detectable and measurable. Usingfluorophore and quencher moieties in this manner, the molecular beaconis only “on” in the “open” conformation and indicates that the probe isbound to the target by emanating an easily detectable signal. Theconformational state of the probe alters the signal generated from theprobe by regulating the interaction between the label moieties. Examplesof donor/acceptor label pairs that may be used in connection with theinvention, include fluorescein/tetramethylrhodamine,EDANS/fluororescein, EDANS/DABCYL, coumarin/DABCYL,fluorescein/fluorescein, BODIPY FL/BODIPY FL, fluorescein/DABCYL,lucifer yellow/DABCYL, BODIPY/DABCYL, eosine/DABCYL, erythrosine/DABCYL,tetramethylrhodamine/DABCYL, Texas Red/DABCYL, CY5/BH1, CY5/BH2,CY3/BH1, CY3/BH2, fluorescein/QSY7, FAM/BHQ1 and Quasar/BHQ1. Thosehaving an ordinary level of skill in the art will understand that whendonor and acceptor dyes are different, energy transfer can be detectedby the appearance of sensitized fluorescence of the acceptor or byquenching of donor fluorescence. When the donor and acceptor species arethe same, energy can be detected by the resulting fluorescencedepolarization. Non-fluorescent acceptors such as DABCYL and the QSY 7dyes advantageously eliminate the potential problem of backgroundfluorescence resulting from direct (i.e., non-sensitized) acceptorexcitation. Preferred fluorophore moieties that can be used as onemember of a donor-acceptor pair include fluorescein, ROX, and the CYdyes (such as CY5).

Synthetic techniques and methods of bonding labels to nucleic acids anddetecting labels are well known in the art (e.g., see Sambrook et al.,Molecular Cloning. A Laboratory Manual. 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; Nelson etal., U.S. Pat. No. 5,658,737; Woodhead et al., U.S. Pat. No. 5,656,207;Hogan et al., U.S. Pat. No. 5,547,842; Arnold et al., U.S. Pat. No.5,283,174; Kourilsky et al., U.S. Pat. No. 4,581,333), and Becker etal., European Patent App. No. 0 747 706.

Probes that hybridize to the amplified sequences include hairpinoligonucleotides such as Molecular Torches and linear oligonucleotidesthat substantially do not form conformations held by intramolecularbonds. Preferably, said probes may include labels. Linear probeembodiments may include a chemiluminescent compound as the label, e.g. achemiluminescent AE compound attached to the probe sequence via a linker(substantially as described in U.S. Pat. Nos. 5,585,481 and 5,639,604,particularly at column 10, line 6 to column 11, line 3, and in Example 8therein). Examples of labeling positions are a central region of theprobe oligomer and near a region of A:T base pairing, at a 3′ or 5′terminus of the oligomer, and at or near a mismatch site with a knownsequence that is not the desired target sequence. Hairpin or linearprobes may be labeled with any of a variety of different types ofinteracting labels, where one interacting member is usually attached tothe 5′ end of the probe and the other interacting member is attached tothe 3′ end of the probe. Dye labeled probes, including dual labeledprobes, single labeled probes, AE labeled probes and the like, aregenerally known. Dual labeled probes can be labeled at one end with afluorescent label (“F”) that absorbs light of a particular wavelength orrange and emits light another emission wavelength or range and at theother end with a quencher (“Q”) that dampens, partially or completely,signal emitted from the excited F when Q is in proximity with thefluorophore. Such a probe may be referred to as labeled with afluorescent/quencher (F/Q) pair. One embodiment of a hairpin probe is a“molecular torch” that detects an amplified product to indicate whethera target sequence is present in the sample after the amplification step.A molecular torch probe comprises a target binding domain and a closingdomain, as is described above. These domains allow the molecular torchto exist in open and closed conformations, depending on whether thetorch is bound to a target. (See also, U.S. Pat. Nos. 6,849,412;6,835,542; 6,534,274; and 6,361,945). Another hairpin probe embodimentis a “molecular beacon” which is generally described in Tyagi et al.,1998, Nature Biotechnol. 16:49-53, and in U.S. Pat. Nos. 5,118,801; and5,312,728. Methods for using such hairpin probes to detect the presenceof a target sequence are well known in the art.

The methods for amplifying a target nucleic acid sequence present in thenucleic acid of Adenovirus can therefore include an optional furtherstep for detecting amplicons. This procedure preferably involves a stepfor contacting a test sample with a hybridization assay probe thatpreferentially hybridizes to the target nucleic acid sequence, or thecomplement thereof, under stringent hybridization conditions, therebyforming a probe:target duplex that is stable for detection. Next thereis a step for determining whether the hybrid is present in the testsample as an indication of the presence or absence of Adenovirus nucleicacids in the test sample. This may involve detecting the probe:targetduplex, and preferably involves homogeneous assay systems.

Hybridization assay probes useful for detecting Adenovirus nucleic acidsequences include a sequence of bases substantially complementary to aAdenovirus target nucleic acid sequence. Thus, probes of the inventionhybridize one strand of a Adenovirus target nucleic acid sequence, orthe complement thereof. These probes may optionally have additionalbases outside of the targeted nucleic acid region which may or may notbe complementary to Adenovirus nucleic acid.

Preferred probes are sufficiently homologous to the target nucleic acidto hybridize under stringent hybridization conditions corresponding toabout 42° C., or more preferably about 60° C. when the saltconcentration is in the range of 0.6-0.9 M. Preferred salts includelithium chloride, but other salts such as sodium chloride and sodiumcitrate also can be used in the hybridization solution. Example highstringency hybridization conditions are alternatively provided by about42° C., or more preferably about 60° C., and 0.48 M sodium phosphatebuffer, 0.1% sodium dodecyl sulfate, and 1 mM each of EDTA and EGTA, orby 0.6 M LiCl, 1% lithium lauryl sulfate, 60 mM lithium succinate and 10mM each of EDTA and EGTA. Probes in accordance with the invention havesequences complementary to, or corresponding to different domains of theAdenovirus genome. Certain probes that are preferred for detectingAdenovirus nucleic acid sequences have a probe sequence, which includesthe target-complementary sequence of bases together with any basesequences that are not complementary to the nucleic acid that is to bedetected, in the length range of from 10-100 nucleotides. Probes fordetecting Adenovirus nucleic acid sequences have target-complementarysequences in the length range of from 15-30, from 16-24, from 18-22 orfrom 18-20 nucleotides. Of course, these target-complementary sequencesmay be linear sequences, or may be contained in the structure of amolecular beacon or other construct having one or more optional nucleicacid sequences that are non-complementary to the Adenovirus targetsequence that is to be detected. As indicated above, probes may be madeof DNA, RNA, a combination DNA and RNA, a nucleic acid analog, orcontain one or more modified nucleosides (e.g., a ribonucleoside havinga 2′-O-methyl substitution to the ribofuranosyl moiety).

Kits

The oligomers for use in the methods described herein are suited forpreparation of kits. Such a kit may comprise containers, each with oneor more of the various oligomers optionally together with one or more ofthe reagents (eg. enzymes) required to perform the methods describedherein. The components of the kit may be supplied in concentrated form.A set of instructions for using the components of the kit will alsotypically be included. Where the kit comprises combinations of oligomersthen the individual oligomers may be provided in individual form, withappropriate instructions for mixing same, or combinations thereof thatare ready mixed.

In one aspect, there is provided a kit comprising the composition of thepresent invention and optionally a set of instructions for performingsame. In one embodiment, this composition comprises a firstamplification oligomer comprising, consisting or consisting essentiallyof a target hybridizing sequence as set forth in SEQ ID NO: 25 or SEQ IDNO: 26 or a combination of two or more thereof in combination with atleast one second amplification oligomer comprising, consisting orconsisting essentially of SEQ ID NO: 27 or SEQ ID NO: 28 or acombination thereof. Accordingly, the composition may comprise SEQ IDNos 25, 26, 27 and 28; SEQ ID Nos 26, 27 and 28; SEQ ID Nos 25, 26 and28; SEQ ID Nos 25, 26 and 27; SEQ ID Nos 25, 27 and 28; SEQ ID Nos 25and 27; SEQ ID Nos 25 and 28; SEQ ID Nos 26 and 27 and SEQ ID Nos 26 and28. According to a further preferred embodiment, the composition maycomprise any one or more of these combinations together with at leastone probe comprising, consisting or consisting essentially of thesequence set forth in SEQ ID No. 36 or SEQ ID No. 37 or a combinationthereof. Thus, for example, a particular preferred combination is any ofSEQ ID Nos 25, 26, 27 and 28; SEQ ID Nos 26, 27 and 28; SEQ ID Nos 25,26 and 28; SEQ ID Nos 25, 26 and 27; SEQ ID Nos 25, 27 and 28; SEQ IDNos 25 and 27; SEQ ID Nos 25 and 28; SEQ ID Nos 26 and 27 and SEQ ID Nos26 and 28 in combination with SEQ ID No. 36 and/or SEQ ID No. 37.

Correlation of Detection of a Target Sequence with Diagnosis

The detection of amplified target sequences characteristic of Adenovirusin a biological sample from an individual is indicative of infection byAdenovirus.

EXAMPLES Example 1 Analysis of Amplification Primers and Probes

Materials & Methods

In a first amplification reaction, the following was used: Fast StartMaster Buffer (Roche) at 1× to 2× concentration, 2 Units of Fast StartTaq DNA polymerase (Roche), 100 nM of a forward amplification primer(SEQ ID No. 5) and 100 nM of a reverse amplification primer (SEQ ID No.6 or SEQ ID No. 8) and 100 nM probe (SEQ ID No. 10).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus added per reaction.Control reactions were performed by setting up a reaction as describedabove but not adding any template nucleic acids. The amplificationcycles used were as follows for both sets of amplification reactions:Hold for 600 seconds at 95 deg. C with optics off; 95 deg. C. for 30seconds with optics off and 55 deg. C. for 60 seconds with optics on (5cycles); 95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60seconds (40 cycles) with optics on.

Results

TABLE 1 Adenovirus Amplification and Detection with Primer and ProbeSets SEQ ID Nos. SEQ ID Nos. 5, 6 and 10 5, 8 and 10 C_(T) RFU C_(T) RFUTarget/Sample 26.9 519 26.4 383

The results are presented as C_(T/)RFU (cycle threshold/relativefluorescent unit) values and represent the average of 12 experimentsusing various Adenovirus serotypes. Amplification was not seen in any ofthe control reactions.

Conclusion

The primers and probes used appeared to be sensitive and specific forAdenovirus nucleic acid.

Example 2 Analysis of Further Amplification Primers and Probes

Materials & Methods

The following reagents were used: Fast Start Master Buffer (Roche) at 1×to 2× concentration, 2 Units of Fast Start Taq DNA polymerase (Roche),200 nM of a forward amplification primer (SEQ ID No. 11 or SEQ ID No.12) and 200 nM of a reverse amplification primer (SEQ ID No. 13 or SEQID No: 15) and 200 nM probe (SEQ ID No. 17 or SEQ ID No. 19).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus added per reaction.Control reactions were performed by setting up a reaction as describedabove but not adding any template nucleic acids. The amplificationcycles used were as follows: Hold for 600 seconds at 95 deg. C withoptics off; 95 deg. C. for 30 seconds with optics off and 55 deg. C. for60 seconds with optics on (5 cycles); 95 deg. C. for 10 seconds withoptics off and 55 deg. C. for 60 seconds (40 cycles) with optics on.

Results

TABLE 2 Adenovirus Amplification and Detection with Primer and ProbeSets C_(T) RFU C_(T) RFU SEQ ID Nos. SEQ ID Nos. 11, 13 and 17 11, 13and 19 Target/Sample 8.7 29.8 32 460 SEQ ID Nos. SEQ ID Nos. 11, 15 and17 11, 15 and 19 Target/Sample 13.3 29.5 32.3 406.4 SEQ ID Nos. SEQ IDNos. 12, 15 and 19 12, 15 and 17 Target/Sample 29.6 620 12.6 32.8 SEQ IDNos. SEQ ID Nos. 12, 13 and 19 12, 13 and 17 Target/Sample 29.6 504 8.318.9

The results are presented as C_(T)/RFU values and represent the averageof 8 experiments using various Adenovirus serotypes. Amplification wasnot seen in any of the control reactions.

Conclusion

Combinations of SEQ ID Nos. 11, 13 and 19, SEQ ID Nos. 11, 15 and 19,SEQ ID Nos. 12, 15 and 19 or SEQ ID Nos. 12, 13 and 19 were sensitiveand specific for Adenovirus nucleic acid. The combinations comprisingthe SEQ ID No. 12 forward primer appears to have better sensitivity thanthe combination comprising the SEQ ID No. 11 forward primer. Thecombination comprising SEQ ID Nos. 12, 15 and 19 appeared to be mostsensitive in these experiments.

Example 3 Adenovirus Serotype Analysis Using SEQ ID Nos. 12, 15 and 19

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 2 Units of Fast Start Taq DNA polymerase (Roche), 400 nMof a forward amplification primer (SEQ ID No. 12) and 400 nM of areverse amplification primer (SEQ ID No. 15) was used together with 400nM probe (SEQ ID No. 19). The total reaction volume was 20 microlitreswith 5 microlitres of template nucleic acid extracted from Adenovirusadded per reaction. Control reactions were set-up, but no templatenucleic acid was added. The amplification cycles used were as follows:Hold for 600 seconds at 95 deg. C with optics off; 95 deg. C. for 30seconds with optics off and 55 deg. C. for 60 seconds with optics on (5cycles); 95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60seconds (40 cycles) with optics on.

Results

TABLE 3 Adenovirus Serotype Analysis Serotype C_(T) RFU  2-1 26 1203 4-1 31 666  6-1 29 926  7-1 32 605  9-1 26 1137 10-1 28 1252 11-1 28630 12-1 26 1119 13-1 26 1100 14-1 30 682 15-1 26 1078 16-1 29 723 17-123 1100 18-1 34 387 19-1 27 1146 20-1 23 996 21-1 31 568 22-1 25 104423-1 23 1109 24-1 25 1221 25-1 32 836 26-1 24 1107 27-1 25 1070 28-1 26989 29-1 27 1116 30-1 22 1166 31-1 21 1127 33-1 28 941 34-1 28 654 35-129 542 36-1 24 997 37-1 24 1125 38-1 26 1033 39-1 23 1143 40-1 27 111441-1 25 994 42-1 23 1125 43-1 22 1149 44-1 22 1141 45-1 27 1071 46-1 271047 47-1 22 1144 48-1 25 1174 49-1 26 1068 50-1 25 672 51-1 26 1099 1-1 29 956  3-1 32 540  5-1 29 791 7A-1  26 632  8-1 34 553 32-2 24 974

The Serotype column is set-up to reflect “serotype number-1×10^(x)TCID₅₀/mL.” C_(T) values have all been rounded down. The results arepresented as C_(T)/RFU values.

Conclusion

The combination of SEQ ID Nos. 12, 15 and 19 was able to detect allserotypes of Adenovirus that were tested.

Example 4 Analysis of Further Probe Combinations Together with SEQ IDNo. 12 and 15 Primers

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 2 Units of Fast Start Taq DNA polymerase (Roche), 100 nMof a forward amplification primer (SEQ ID No. 12) and 100 nM of areverse amplification primer (SEQ ID No. 15) was used together witheither: 150 nM probe (SEQ ID No. 21) and 50 nM probe (SEQ ID No. 24);100 nM probe (SEQ ID No. 21) and 100 nM probe (SEQ ID No. 24); 50 nMprobe (SEQ ID No. 21) and 150 nM probe (SEQ ID No. 24). The totalreaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from Adenovirus added per reaction. Controlreactions were set-up without the addition of template nucleic acid. Theamplification cycles used were as follows: Hold for 600 seconds at 95deg. C with optics off; 95 deg. C. for 30 seconds with optics off and 55deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for 10seconds with optics off and 55 deg. C. for 60 seconds (40 cycles) withoptics on.

Results

TABLE 4 Amplification and detection using different concentrations ofprobe combinations 150 nM SEQ ID 100 nM SEQ ID No. 21 and 50 nM No. 21and 100 nM SEQ ID No. 24; SEQ ID No. 24; C_(T) RFU C_(T) RFU Target 34.8291 26.9 318 50 nM SEQ ID No. 21 and 150 nM SEQ ID No. 24 C_(T) RFUTarget 26.8 339

The results are presented as C_(T)/RFU values and represent the averageof 6 experiments using various Adenovirus serotypes.

Conclusion

SEQ ID No. 21 and ID No. 24 probes in combination with SEQ ID No. 12 and15 were able to sensitively and specifically detect Adenovirus at thevarious concentrations tested.

Example 5 Analysis of Further Probe and Primer Combinations for theDetection of Adenovirus

Materials & Methods

The following reagents used:

Fast Start Master Buffer (Roche) at 1× concentration, 2 Units of FastStart Taq DNA polymerase (Roche) and either: (i) 50 mM of a forwardamplification primer (SEQ ID No. 25), 50 mM of a forward amplificationprimer (SEQ ID No. 26), 50 mM of a reverse amplification primer (SEQ IDNo. 27), 50 mM of a reverse amplification primer (SEQ ID No. 28) and 100nM of probes (SEQ ID No. 21 and SEQ ID No. 23); (ii) 50 mM of a forwardamplification primer (SEQ ID No. 26), 50 mM of a reverse amplificationprimer (SEQ ID No. 27), 50 mM of a reverse amplification primer (SEQ IDNo. 28) and 100 nM of probes (SEQ ID No. 21 and SEQ ID No. 23); (iii) 50mM of a forward amplification primer (SEQ ID No. 25), 50 mM of a reverseamplification primer (SEQ ID No. 27), 50 mM of a reverse amplificationprimer (SEQ ID No. 28) and 100 nM of probes (SEQ ID No. 21 and SEQ IDNo. 23); (iv) 50 mM of a forward amplification primer (SEQ ID No. 25),50 mM of a forward amplification primer (SEQ ID No. 26), 50 mM of areverse amplification primer (SEQ ID No. 28) and 100 nM of probes (SEQID No. 21 and SEQ ID No. 23); (v) 50 mM of a forward amplificationprimer (SEQ ID No. 25), 50 mM of a forward amplification primer (SEQ IDNo. 26), 50 mM of a reverse amplification primer (SEQ ID No. 28) and 100nM of probes (SEQ ID No. 21 and SEQ ID No. 23); (vi) 50 mM of a forwardamplification primer (SEQ ID No. 25), 50 mM of a forward amplificationprimer (SEQ ID No. 26), 50 mM of a reverse amplification primer (SEQ IDNo. 27), 50 mM of a reverse amplification primer (SEQ ID No. 28) and 100nM of probe (SEQ ID No. 23); or (vii) 50 mM of a forward amplificationprimer (SEQ ID No. 25), 50 mM of a forward amplification primer (SEQ IDNo. 26), 50 mM of a reverse amplification primer (SEQ ID No. 27), 50 mMof a reverse amplification primer (SEQ ID No. 28) and 100 nM of probes(SEQ ID No. 21).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus added per reaction. Twodifferent concentrations were tested.

The amplification cycles used were as follows: Hold for 600 seconds at95 deg. C with optics off; 95 deg. C. for 30 seconds with optics off and55 deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for 10seconds with optics off and 55 deg. C. for 60 seconds (40 cycles) withoptics on.

Results

Tables 5a-5d. Amplification and detection using different concentrationsand combinations of primers and probes.

TABLE 5a SEQ ID Nos. 25, 26, SEQ ID Nos. 26, 27, 28, 21 and 23 27, 28,21 and 23 C_(T) RFU C_(T) RFU Target (10¹) 38.5 240 39.2 143 Target(10³) 29.8 373 30.8 227

TABLE 5b SEQ ID Nos. 25, SEQ ID Nos. 25, 27, 28, 21 and 23 26, 28, 21and 23 C_(T) RFU C_(T) RFU Target (10¹) 37.8 212 41.5 99 Target (10³)30.2 275 32 258

TABLE 5c SEQ ID Nos. 25, SEQ ID Nos. 25, 26, 27, 21 and 23 26, 27, 28and 23 C_(T) RFU C_(T) RFU Target (10¹) 41.8 96 37.7 254 Target (10³)31.8 320 30 360

TABLE 5d SEQ ID Nos. 25, 26, 27, 28 and 21 C_(T) RFU Target (10¹) 7.1 21Target (10³) 0 3

The results are presented as RFU values and represent the average of 6experiments for each concentration.

Conclusion

Leaving out one of the primers or probes from the assay made littledifference for the most part. However, omitting probe SEQ ID No. 23resulted in lower detection in this particular experiment.

Example 6 Analysis of Primer and Probe Combinations for DetectingAdenovirus 18

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche), 150 nMforward amplification primers (SEQ ID No. 25 and SEQ ID No. 26) and 150nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 300 nM probe (SEQ ID No. 29).

The total reaction volume was 20 microlitres with 5 microlitres oftemplate nucleic acid extracted from Adenovirus 18 added per reaction.The amplification cycles used were as follows: Hold for 600 seconds at95 deg. C with optics off; 95 deg. C. for 30 seconds with optics off and55 deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for 10seconds with optics off and 55 deg. C. for 60 seconds (40 cycles) withoptics on.

Results

TABLE 6 Amplification and detection of Adenovirus 18. Serotype C_(T) RFU18-6 17 1240 18-5 20 975 18-4 24 1242 18-3 30 1023 18-2 33 942 18-1 35747 18-0 31 1215

The Serotype column is set-up to reflect “serotype number-1×10^(x)TCID₅₀/mL.” C_(T) values have all been rounded down. The results arepresented as C_(T)/RFU values.

Conclusion

This combination of primers and probes successfully detects Adenovirus18.

Example 7 Analysis of Further Primer and Probe Combinations forDetecting Adenovirus

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche), 150 nMforward amplification primers (SEQ ID No. 31 and SEQ ID No. 26) and 150nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 150 nM probe (SEQ ID No. 21 and SEQ ID No. 23). Thetotal reaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from various Adenovirus serotypes added perreaction. The amplification cycles used were as follows: Hold for 600seconds at 95 deg. C with optics off; 95 deg. C. for 30 seconds withoptics off and 55 deg. C. for 60 seconds with optics on (5 cycles); 95deg. C. for 10 seconds with optics off and 55 deg. C. for 60 seconds (40cycles) with optics on.

Results

TABLE 7 Primer and probe combinations for detecting various Adenovirusserotypes. FAM Cy5 Serotype C_(T) RFU C_(T) RFU 1 38.7 40 35.9 172 337.4 59 35.5 208 4 0 22 37.2 185 7 32.2 711 32 209 11 24.2 843 32.2 19014 28.8 737 31.9 196 16 23.8 879 32.1 212 21 31.9 671 31.4 219 25 32.8399 31.7 217 34 29.3 645 31.7 205 35 29.6 771 30.7 220 50 24.6 786 30.9210

The results are presented as C_(T/)RFU values. The Fam-channel showsdetection results for the template nucleic acids. The Cy5-channel showsdetection results for an internal control nucleic acid.

Conclusion

With the exception of serotype 4, this combination of primers and probessuccessfully detected all of the serotypes tested.

Example 8 Analysis of Further Primer and Probe Combinations forDetecting Adenovirus

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche) andeither: (i) 150 nM forward amplification primers (SEQ ID Nos. 33 and 34)and 150 nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No.28) were used together with 150 nM probe (SEQ ID No. 21 and SEQ ID No.23); (ii) 150 nM forward amplification primers (SEQ ID Nos. 33 and 35)and 150 nM reverse amplification primers (SEQ ID No. 27 and SEQ ID No.28) were used together with 150 nM probe (SEQ ID No. 21 and SEQ ID No.23); or (iii) 150 nM forward amplification primers (SEQ ID Nos. 34 and35) and 150 nM reverse amplification primers (SEQ ID No. 27 and SEQ IDNo. 28) were used together with 150 nM probe (SEQ ID No. 21 and SEQ IDNo. 23). The total reaction volume was 20 microlitres with 5 microlitresof template nucleic acid extracted from various Adenovirus serotypesadded per reaction. The amplification cycles used were as follows: Holdfor 600 seconds at 95 deg. C with optics off; 95 deg. C. for 30 secondswith optics off and 55 deg. C. for 60 seconds with optics on (5 cycles);95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60 seconds(40 cycles) with optics on.

Results

Tables 8a-8c. Amplification and detection of various Adenovirusserotypes using combinations of primers and probes.

TABLE 8a SEQ ID Nos. 33, 34, 27, 28, 21 and 23 FAM Cy5 Serotype C_(T)RFU C_(T) RFU 1 35.6 318 35.9 243 3 37.3 87 35.8 210 4 36.9 150 35.6 23219 38.1 95 35.2 217 31 35.7 247 34.6 257 41 36.7 244 35.9 285 14 29.7868 31.5 250

TABLE 8b SEQ ID Nos. 33, 35, 27, 28, 21 and 23 FAM Cy5 Serotype C_(T)RFU C_(T) RFU 1 0 12 35.7 248 3 36.4 159 35.1 231 4 36.8 171 35.6 249 1936.7 151 35.3 181 31 35.8 170 34.6 197 41 39.3 50 36 243 14 29.2 106231.5 256

TABLE 8c SEQ ID Nos. 34, 35, 27, 28, 21 and 23 FAM Cy5 Serotype C_(T)RFU C_(T) RFU 1 37.5 198 35.5 199 3 0 5 35.5 213 4 0 15 36.1 158 19 0 1235.1 224 31 35.6 369 34.6 240 41 36.6 284 35.7 263 14 33.1 942 32.1 203

The results are presented as C_(T) and RFU values. The Fam-channel showsdetection results for the template nucleic acids. The Cy5-channel showsdetection results for an internal control nucleic acid.

Conclusion

Table 8a of primers and probes successfully detected all of theserotypes tested. Tables 8b and 8c detected most serotypes tested.

Example 9 Analysis of Further Primer and Probe Combinations forDetecting Adenovirus

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche) and 150nM forward amplification primers (SEQ ID Nos. 25 and 26) and 150 nMreverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 150 nM probe (SEQ ID No. 36 and SEQ ID No. 37). Thetotal reaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from the Adenovirus 19 serotype positive controlplasmid, which added per reaction at six different concentrations. Theamplification cycles used were as follows: Hold for 600 seconds at 95deg. C with optics off; 95 deg. C. for 30 seconds with optics off and 55deg. C. for 60 seconds with optics on (5 cycles); 95 deg. C. for 10seconds with optics off and 55 deg. C. for 60 seconds (40 cycles) withoptics on.

Results

TABLE 9 Amplification and detection of a serial dilution of targetnucleic acid FAM Concentration C_(T) RFU 10⁴ 28.1 1127 10⁴ 28.2 1040 10⁴28 1196 10³ 31.9 938 10³ 32.1 922 10³ 32.3 969 10² 35.3 865 10² 35.4 80010² 35.2 800 10¹ 37.8 571 10¹ 33.6 59 10¹ 38.6 419 10⁰ 0 10 10⁰ 0 0 10⁰0 0 10⁻¹ 0 0 10⁻¹ 0 0 10⁻¹ 0 0

The results are presented as C_(T/)RFU values.

Conclusion

These primers and probes successfully detected the control testedAdenovirus 19 serotype.

Example 10 Further Analysis of the Primer and Probe Combination fromExample 9

Materials & Methods

The following reagents used: Fast Start Master Buffer (Roche) at 1×concentration, 3 Units of Fast Start Taq DNA polymerase (Roche) and 150nM forward amplification primers (SEQ ID Nos. 25 and 26) and 150 nMreverse amplification primers (SEQ ID No. 27 and SEQ ID No. 28) wereused together with 150 nM probe (SEQ ID No. 36 and SEQ ID No. 37). Thetotal reaction volume was 20 microlitres with 5 microlitres of templatenucleic acid extracted from various Adenovirus serotypes and tested at aconcentration of 3×10⁰. The amplification cycles used were as follows:Hold for 600 seconds at 95 deg. C with optics off; 95 deg. C. for 30seconds with optics off and 55 deg. C. for 60 seconds with optics on (5cycles); 95 deg. C. for 10 seconds with optics off and 55 deg. C. for 60seconds (40 cycles) with optics on.

Results

TABLE 10 Amplification and detection of Adenovirus target nucleic acidsSerotype C_(T) RFU 2 31.6 1465 5 33.9 903 6 0 0 7 39 366 8 37.4 625 9 321223 10 36.3 837 11 26.6 950 12 31.8 1176 13 29.7 1487 14 34 671 15 33.51018 16 33.4 729 17 30.2 1622 18 40 175 20 27.2 1217 21 34.1 733 22 31.21150 23 30.1 1471 24 33.2 1110 25 37.3 661 26 29.1 1814 27 29.5 1637 2834.2 1032 29 32.9 1159 30 28.4 1496 32 26.6 2079 33 33.9 1017 34 34.2690 35 33.1 702 36 29.1 1312 37 30.2 1393 38 31.8 1202 39 30 1650 4032.6 1290 42 29.1 1261 43 28.8 1832 44 23.3 1218 45 32.1 1289 46 32.51183 47 26.4 1209 48 29.4 1565 49 31.5 1264 50 32 846 51 31.4 1125

The results are presented as C_(T) and RFU values.

Discussion

All of the serotypes tested were detected using this primer and probeconcentration with the exception of serotype 6. This serotype wassuccessfully detected at 3×10¹ TCID₅₀/mL and above.

Example 11 Exemplary Nucleic Acid Sequences

The instant example provides exemplary sequences that are useful withthe present invention. This table does not limit the scope of theinvention. Sequences are presented according to World IntellectualProperty Organization (WIPO) Handbook on Industrial Property Informationand Documentation, Standard ST.25 (1998), including Tables 1 through 6of Appendix 2.

TABLE 11 Exemplary nucleic acid sequences SEQ ID No Sequence 5′ --> 3′ 1 CAGGACGCCTCGGRGTAYCTSAG  2 GGAGCCACVGTGGGRTT  3 AAYCCCACBGTGGCTCC  4CCGGGTCTGGTGCAGTTTGCCCGC  5 CACATCGCCGGACAGGA  6 CATACTGAAGTAGGTGTCTGT 7 ACAGACACCTACTTCAGTATG  8 CGGTGGTCACATCGTGG  9 CCACGATGTGACCACCG 10AGTACCTCAGTCCGGGTCTGGTG 11 ATGGCTACCCCTTCGATG 12 ACCCCMTCGATGATGCC 13GCGGGCGAATTGCACCA 14 TGGTGCAATTCGCCCGC 15 GCGGGCAAAYTGCACCA 16TGGTGCARTTTGCCCGC 17 GACTCAGGTACTCCGAAGCATCCT 18AGGATGCTTCGGAGTACCTGAGTC 19 CTCAGGTACTCCGAGGCGTCCT 20AGGACGCCTCGGAGTACCTGAG 21 CTCAGGTACTCCGAAGCATCCT 22AGGATGCTTCGGAGTACCTGAG 23 CAGGTACTCCGAGGCGTCCT 24 AGGACGCCTCGGAGTACCTG25 ACCCCATCGATGATGCC 26 ACCCCCTCGATGATGCC 27 GCGGGCAAACTGCACCA 28GCGGGCAAATTGCACCA 29 CTCAGGTATTCCGAGGCATCCT 30 AGGATGCCTCGGAATACCTGAG 31ACCCCATCGATGCTGCC 32 ACCCCATCGATGATGCC 33 TGGGCGTACATGCACATC 34GTGGTCTTACATGCACATC 35 GTGGGCATACATGCACATC 36 AGGATGCTTCGGAGTACCTGAG 37AGGACGCCTCGGAGTACCTG 38 ARTGGKCDTACATGCACATC 39 CAGGACGCCTCGGAGTACCT 40AGGATGCTTCGGAGTACCTGAG 41 CACGATGTGACCACAGA 42 CAYGATGTGACCACAGA 43CACGAYGTGACCACAGA 44 CACGATGTGACCACSGA 45 CACGATGTGACCACVGA 46CAYGAYGTGACCACVGAHuman adenovirus 9 gene for hexon, complete cds, strain: Hicks.GenBank Accession Number AB330090.1 and gi number GI:190356540.First seen at NCBI on Jun. 13 2008. (SEQ ID NO: 47)atggccaccccctcgatgatgccgcagtgggcgtacatgcacatcgccgggcaggacgcctcggagtacctgagcccgggtctggtgcagtttgcccgcgccaccgacacgtacttcagcctgggcaacaagtttaggaaccccacggtggccccgacccacgatgtgaccacggaccggtcccagcgtctgacgctgcgcttcgtgcccgtggatcgcgaggacaccacgtactcgtacaaggcgcgcttcactctggccgtgggcgacaaccgggtgctagacatggccagcacttactttgacatccgcggcgtcctggaccgcggtcccagcttcaaaccctactcgggcacagcttacaacagtctggcccccaagggtgcccccaactccagccagtggcttgcaaaagacaccaatgctggcgatcaagcattaaaaacccacacacatggcgtagctgctatggggggaacagatatcacagcaaagggtttgcaaattggtgttgacacgactgaaaacaagaatgagcctatttatgcaaatgaaatataccagccagaacctcaggtaggagaggaaaacttgcaagatgttgaaaacttttatggaggcagagctcttaaaaaagaaaccaaaatgaaaccttgctatggctcgtttgccagacccacaaatgaaaaaggcggtcaagccaaatttttaactgacggcgatggtcagctaactaaaaatcatgatatcacaatgaatttctttgacactcctggaggaacagttggtcaggatactgaacttgaagcagacattgttatgtatgctgagaatgtgcatctggaaactccagacacgcatgtggtgtacaaaccaggaacttctgatgagagttcagaagcaaatttggttcagcagtccatgccaaacaggcccaactacatcggcttcagggacaactttgtgggtctcatgtactataacagcactggcaacatgggtgtgctggctggtcaagcatctcagttgaatgctgtggtcgacttgcaagacagaaacacagagctgtcttaccagctcttgctagattctctgggtgacagaaccagatactttagcatgtggaactctgcagtggacagttatgatcctgatgtcaggattattgaaaatcacggtgtggaagatgaacttccaaactattgcttcccattggatggagctggcactaatgctacctaccaaggtgtaaaagttaaaaatggccaagatggagatgtaaacgcagattgggaaaaagatccaaatcttgcttcacgaaaccaaatatgcaagggtaacatcttcgccatggagatcaacctccaggccaacctgtggaagagttttctgtactcgaatgtggccctgtacctgcccgactcatacaagtacacgccggccaacgtcacgctgcccgccaataccaacacctacgagtacatgaacggccgcgtggtagccccctcgctggtggacgcctacatcaacatcggcgcccggtggtcgctggaccccatggacaacgtcaacccattcaaccaccaccgcaacgcgggcctgcgttaccgctccatgcttctgggcaacggccgctacgtgcccttccacatccaagtgccccaaaagttctttgccatcaagaacctgctcctgctccccggctcctacacctacgagtggaacttccgcaaggatgtcaacatgatcctgcagagttccctcggaaacgacctgcgcgtcgacggcgcctccgtccgcttcgacagcgtcaacctctacgccacattcttccccatggcgcacaacaccgcctccaccctggaagccatgctgcgcaacgacaccaacgaccagtccttcaacgactacctctcggccgccaacatgctctaccccatcccggccaaggccaccaacgtgcccatctccatcccctcgcgcaactgggccgccttccgcggctggagtttcacccggctcaagaccaaagaaactccctccctcggctcgggtttcgatccctactttgtatactcgggttccatcccctacctcgacgggaccttctacctcaaccacaccttcaagaaggtctccatcatgttcgactcctcggtcagctggcccggcaacgaccggctgctcacgccgaacgagttcgagatcaagcgcagtgtcgacggggagggctacaatgtggcccaatgcaacatgaccaaggactggttcctcgtccagatgctctcccactacaacatcggctaccagggcttccacgtgcccgagggctacaaggaccgcatgtactccttcttccgcaacttccagcccatgagcaggcaggtggtcgatgagatcaactacaaggactacaaggccgtcaccctgcccttccagcacaacaactcgggcttcaccggctaccttgcacccaccatgcgtcaggggcagccctaccccgccaacttcccctatcctctcatcggccagacagccgtgccctctgtcacccagaaaaagttcctctgcgacagggtcatgtggcgcatccccttctccagcaacttcatgtccatgggcgccctcaccgacctgggtcagaacatgctctatgccaactcggcccacgcgctcgacatgaccttcgaggtggaccccatggatgagcccaccctcctctatcttctcttcgaagttttcgacgtggtcagagtgcaccagccgcaccgcggcgtcatcgaggccgtctacctgcgcacgcccttctccgccggcaacgccaccacctaa

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

The invention claimed is:
 1. A composition for use in an Adenovirustarget nucleic acid amplification assay comprising (I) at least threeamplification oligomers capable of stably hybridizing to Adenovirustarget nucleic acid to generate an amplicon during an amplificationreaction, wherein the at least three amplification oligomers are (a) afirst amplification oligomer consisting of a target hybridizing sequenceas set forth in SEQ ID NO:27, (b) a second amplification oligomerconsisting of a target hybridizing sequence as set forth in SEQ IDNO:28, and (c) a third amplification oligomer consisting of targethybridizing sequences as set forth in (i) SEQ ID NO:25 and/or SEQ IDNO:26, (ii) SEQ ID NO:33 and/or SEQ ID NO:34, or (iii) SEQ ID NO:31and/or SEQ ID NO:26; and (II) at least one labelled detection probeoligonucleotide configured to specifically hybridize to a portion ofsaid amplicon.
 2. The composition of claim 1, wherein the at least threeamplification oligomers are selected from the group consisting of SEQ IDNos 25, 26, 27 and 28; SEQ ID Nos 26, 27 and 28; SEQ ID Nos 27, 28, 33and 34; SEQ ID Nos 26, 27, 28 and 31; and SEQ ID Nos 25, 27 and
 28. 3. Areaction mixture for use in an Adenovirus detection assay, wherein thereaction mixture comprises (I) at least three amplification oligomerscapable of stably hybridizing to Adenovirus target nucleic acid togenerate an amplicon during an amplification reaction, wherein the atleast three amplification oligomers are (a) a first amplificationoligomer consisting of a target hybridizing sequence as set forth in SEQID NO:27, (b) a second amplification oligomer consisting of a targethybridizing sequence as set forth in SEQ ID NO:28, and (c) a thirdamplification oligomer consisting of target hybridizing sequences as setforth in (i) SEQ ID NO:25 and/or SEQ ID NO:26, (ii) SEQ ID NO:33 and/orSEQ ID NO:34, or (iii) SEQ ID NO:31 and/or SEQ ID NO:26; and (II) atleast one labelled detection probe oligonucleotide configured tospecifically hybridize to a portion of said amplicon.
 4. The reactionmixture of claim 3, further comprising one or more of a polymerase, afourth amplification oligonucleotide, glycerol, magnesium chloride or adNTP.
 5. The reaction mixture of claim 3, wherein the at least threeamplification oligomer combinations are: amplification oligomerscomprising the target hybridizing sequences consisting of SEQ ID Nos 25,26, 27 and 28, amplification oligomers comprising the target hybridizingsequences consisting of SEQ ID Nos 27, 28, 33 and 34, or amplificationoligomers comprising the target hybridizing sequences consisting of SEQID Nos 26, 27, 28 and
 31. 6. The reaction mixture of claim 3, whereinthe at least one labelled detection probe oligonucleotide comprises atarget hybridizing sequences selected from the group consisting of SEQID Nos 17 to 24, 29, 30, 36, 37, 39 and
 40. 7. The reaction mixture ofclaim 3, wherein the at least one labelled detection probeoligonucleotide is two or more labelled detection probe oligonucleotideseach comprising a target hybridizing sequences selected from the groupconsisting of SEQ ID Nos 17 to 24, 29, 30, 36, 37, 39 and
 40. 8. Thereaction mixture of claim 7, wherein the at least one labelled detectionprobe oligonucleotide is two labelled detection probe oligonucleotidescomprising for the first labelled detection probe oligonucleotide thetarget hybridizing sequence consisting of SEQ ID No 36, and comprisingfor the second labelled detection probe oligonucleotide the targethybridizing sequence consisting of SEQ ID No
 37. 9. The composition ofclaim 1, wherein the at least one labelled detection probeoligonucleotide comprises a target hybridizing sequences selected fromthe group consisting of SEQ ID Nos 17 to 24, 29, 30, 36, 37, 39 and 40.10. The composition of claim 9, wherein the at least one labelleddetection probe oligonucleotide is two or more labelled detection probeoligonucleotides each comprising a target hybridizing sequences selectedfrom the group consisting of SEQ ID Nos 17 to 24, 29, 30, 36, 37, 39 and40.
 11. The composition of claim 10, wherein the at least one labelleddetection probe oligonucleotide is two labelled detection probeoligonucleotides comprising for the first labelled detection probeoligonucleotide the target hybridizing sequence consisting of SEQ ID No36, and comprising for the second labelled detection probeoligonucleotide the target hybridizing sequence consisting of SEQ ID No37.